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In people living with HIV, Kaposi Sarcoma (KS), a vascular neoplasm caused by KS herpesvirus (KSHV/HHV-8), remains one of the most common malignancies worldwide. Individuals living with HIV, receiving otherwise effective antiretroviral therapy, may present with extensive disease requiring chemotherapy. Hence, new therapeutic approaches are needed. The Wilms' tumor 1 (WT1) protein is overexpressed and associated with poor prognosis in several hematologic and solid malignancies and has shown promise as an immunotherapeutic target. We found that WT1 was overexpressed in >90% of a total 333 KS biopsies, as determined by immunohistochemistry and image analysis. Our largest cohort from ACTG, consisting of 294 cases was further analyzed demonstrating higher WT1 expression was associated with more advanced histopathologic subtypes. There was a positive correlation between the proportion of infected cells within KS tissues, assessed by expression of the KSHV-encoded latency-associated nuclear antigen (LANA), and WT1 positivity. Areas with high WT1 expression showed sparse T-cell infiltrates, consistent with an immune evasive tumor microenvironment. We show that major oncogenic isoforms of WT1 are overexpressed in primary KS tissue and observed WT1 upregulation upon de novo infection of endothelial cells with KSHV. KSHV latent viral FLICE-inhibitory protein (vFLIP) upregulated total and major isoforms of WT1, but upregulation was not seen after expression of mutant vFLIP that is unable to bind IKKƴ and induce NFκB. siRNA targeting of WT1 in latent KSHV infection resulted in decreased total cell number and pAKT, BCL2 and LANA protein expression. Finally, we show that ESK-1, a T cell receptor-like monoclonal antibody that recognizes WT1 peptides presented on MHC HLA-A0201, demonstrates increased binding to endothelial cells after KSHV infection or induction of vFLIP expression. We propose that oncogenic isoforms of WT1 are upregulated by KSHV to promote tumorigenesis and immunotherapy directed against WT1 may be an approach for KS treatment.

Drug resistance remains a major obstacle to malaria control and eradication efforts, necessitating the development of novel therapeutic strategies to treat this disease. Drug combinations based on collateral sensitivity, wherein resistance to one drug causes increased sensitivity to the partner drug, have been proposed as an evolutionary strategy to suppress the emergence of resistance in pathogen populations. In this study, we explore collateral sensitivity between compounds targeting the Plasmodium dihydroorotate dehydrogenase (DHODH). We profiled the cross-resistance and collateral sensitivity phenotypes of several DHODH mutant lines to a diverse panel of DHODH inhibitors. We focus on one compound, TCMDC-125334, which was active against all mutant lines tested, including the DHODH C276Y line, which arose in selections with the clinical candidate DSM265. In six selections with TCMDC-125334, the most common mechanism of resistance to this compound was copy number variation of the dhodh locus, although we did identify one mutation, DHODH I263S, which conferred resistance to TCMDC-125334 but not DSM265. We found that selection of the DHODH C276Y mutant with TCMDC-125334 yielded additional genetic changes in the dhodh locus. These double mutant parasites exhibited decreased sensitivity to TCMDC-125334 and were highly resistant to DSM265. Finally, we tested whether collateral sensitivity could be exploited to suppress the emergence of resistance in the context of combination treatment by exposing wildtype parasites to both DSM265 and TCMDC-125334 simultaneously. This selected for parasites with a DHODH V532A mutation which were cross-resistant to both compounds and were as fit as the wildtype parent in vitro. The emergence of these cross-resistant, evolutionarily fit parasites highlights the mutational flexibility of the DHODH enzyme.

PURPOSE: To compare the evaluation metrics for deep learning methods that were developed using imbalanced imaging data in osteoarthritis studies. MATERIALS AND METHODS: This retrospective study utilized 2996 sagittal intermediate-weighted fat-suppressed knee MRIs with MRI Osteoarthritis Knee Score readings from 2467 participants in the Osteoarthritis Initiative study. We obtained probabilities of the presence of bone marrow lesions (BMLs) from MRIs in the testing dataset at the sub-region (15 sub-regions), compartment, and whole-knee levels based on the trained deep learning models. We compared different evaluation metrics (e.g., receiver operating characteristic (ROC) and precision-recall (PR) curves) in the testing dataset with various class ratios (presence of BMLs vs. absence of BMLs) at these three data levels to assess the models performance. RESULTS: In a subregion with an extremely high imbalance ratio, the model achieved a ROC-AUC of 0.84, a PR-AUC of 0.10, a sensitivity of 0, and a specificity of 1. CONCLUSION: The commonly used ROC curve is not sufficiently informative, especially in the case of imbalanced data. We provide the following practical suggestions based on our data analysis: 1) ROC-AUC is recommended for balanced data, 2) PR-AUC should be used for moderately imbalanced data (i.e., when the proportion of the minor class is above 5% and less than 50%), and 3) for severely imbalanced data (i.e., when the proportion of the minor class is below 5%), it is not practical to apply a deep learning model, even with the application of techniques addressing imbalanced data issues.

PurposeGiven the increasing availability of radiation therapy in sub-Saharan Africa, clinical trials that include radiation therapy are likely to grow. Ensuring appropriate delivery of radiation therapy through rigorous quality assurance is an important component of clinical trial execution. We reviewed the process for credentialing radiation therapy sites and radiation therapy quality assurance through the Imaging and Radiation Oncology Core (IROC) Houston Quality Assurance Center for AIDS Malignancy Consortium (AMC)-081, a multicenter study of cisplatin and radiation therapy for women with locally advanced cervical cancer living with HIV, conducted by the AIDS Malignancy Consortium at 2 sites in South Africa and Zimbabwe.Methods and materialsWomen living with HIV with newly diagnosed stage IB2, IIA (>4 cm), IIB-IVA cervical carcinoma (per the 2009 International Federation of Gynecology and Obstetrics [FIGO] staging classifications) were enrolled in AMC-081. They received 3-dimensional conformal external beam radiation therapy (EBRT) to the pelvis (41.4-45 Gy) using a linear accelerator, high-dose-rate brachytherapy (6-9 Gy to point A with each fraction and up to 4 fractions), and concurrent weekly cisplatin (40 mg/m2). IROC reviewed EBRT and brachytherapy quality assurance records after treatment.ResultsAll of the 38 women enrolled in AMC-081 received ±5% of the protocol-specified prescribed dose of EBRT. Geometry of brachytherapy applicator placement was scored as per protocol in all implants. Doses to points A and B, International Commission on Radiation Units and Measurements (ICRU) bladder, or ICRU rectum required correction by IROC in >50% of the implants. In the final evaluation, 58% of participants (n = 22) were treated per protocol, 40% (n = 15) had minor protocol deviations, and 3% (n = 1) had major protocol deviations. No records were received within 60 days of treatment completion as requested in the protocol.ConclusionsMajor radiation therapy deviations were low, but timely submission of radiation therapy data did not occur. Future studies, especially those that include specialized radiation therapy techniques such as stereotactic or intensity-modulated radiation therapy, will require pathways to ensure timely and adequate quality assurance.

Identifying how small molecules act to kill malaria parasites can lead to new chemically validated targets. By pressuring Plasmodium falciparum asexual blood stage parasites with three novel structurally-unrelated antimalarial compounds (MMV665924, MMV019719 and MMV897615), and performing whole-genome sequence analysis on resistant parasite lines, we identify multiple mutations in the P. falciparum acyl-CoA synthetase (ACS) genes PfACS10 (PF3D7_0525100, M300I, A268D/V, F427L) and PfACS11 (PF3D7_1238800, F387V, D648Y, and E668K). Allelic replacement and thermal proteome profiling validates PfACS10 as a target of these compounds. We demonstrate that this protein is essential for parasite growth by conditional knockdown and observe increased compound susceptibility upon reduced expression. Inhibition of PfACS10 leads to a reduction in triacylglycerols and a buildup of its lipid precursors, providing key insights into its function. Analysis of the PfACS11 gene and its mutations point to a role in mediating resistance via decreased protein stability.

BACKGROUND: We performed an integrated biomarker evaluation in pembrolizumab-treated patients with R/M HNSCC enrolled in KEYNOTE-012 or KEYNOTE-055. The relationship between biomarkers and HPV status was explored. METHODS: We evaluated PD-L1 (combined positive score [CPS]), TMB, T-cell-inflamed gene expression profile (Tcellinf GEP), and HPV status. Associations between biomarkers were evaluated by logistic regression (ORR) and Cox regression (PFS, OS). RESULTS: Two hundred and fifty-seven patients (KEYNOTE-012, n = 106; KEYNOTE-055, n = 151) had TMB data available; of these, 254 had PD-L1 and 236 had Tcellinf GEP. TMB, PD-L1, and Tcellinf GEP were each significantly associated with ORR (p

BackgroundFour large community-randomized trials examining universal testing and treatment (UTT) to reduce HIV transmission were conducted between 2012-2018 in Botswana, Kenya, Uganda, Zambia and South Africa. In 2014, the UNAIDS 90-90-90 targets were adopted as a useful metric to monitor coverage. We systematically review the approaches used by the trials to measure intervention delivery, and estimate coverage against the 90-90-90 targets. We aim to provide in-depth understanding of the background contexts and complexities that affect estimation of population-level coverage related to the 90-90-90 targets.MethodsEstimates were based predominantly on "process" data obtained during delivery of the interventions which included a combination of home-based and community-based services. Cascade coverage data included routine electronic health records, self-reported data, survey data, and active ascertainment of HIV viral load measurements in the field.ResultsThe estimated total adult populations of trial intervention communities included in this study ranged from 4,290 (TasP) to 142,250 (Zambian PopART Arm-B). The estimated total numbers of PLHIV ranged from 1,283 (TasP) to 20,541 (Zambian PopART Arm-B). By the end of intervention delivery, the first-90 target (knowledge of HIV status among all PLHIV) was met by all the trials (89.2%-94.0%). Three of the four trials also achieved the second- and third-90 targets, and viral suppression in BCPP and SEARCH exceeded the UNAIDS target of 73%, while viral suppression in the Zambian PopART Arm-A and B communities was within a small margin (~ 3%) of the target.ConclusionsAll four UTT trials aimed to implement wide-scale testing and treatment for HIV prevention at population level and showed substantial increases in testing and treatment for HIV in the intervention communities. This study has not uncovered any one estimation approach which is superior, rather that several approaches are available and researchers or policy makers seeking to measure coverage should reflect on background contexts and complexities that affect estimation of population-level coverage in their specific settings. All four trials surpassed UNAIDS targets for universal testing in their intervention communities ahead of the 2020 milestone. All but one of the trials also achieved the 90-90 targets for treatment and viral suppression. UTT is a realistic option to achieve 95-95-95 by 2030 and fast-track the end of the HIV epidemic.

BackgroundPrevious studies have observed that haemoglobin concentrations can be affected by type of blood collection, analysis methods and device, and that near-in-time population-based surveys report substantially different anaemia prevalence. We investigated whether differences in mean haemoglobin or prevalence of anaemia between near-in-time surveys of the same population were associated with differences in type of blood collection or analytic approach to haemoglobin measurement.MethodsWe systematically identified pairs of population-based surveys that measured haemoglobin in the same population of women of reproductive age (WRA) or preschool-aged children (PSC). Surveys were matched on geographic coverage, urban/rural place of residence, inclusion of pregnant women, time of data collection (within 18 months), and, to the extent feasible, age range. Differences in anaemia prevalence were presented graphically. Random-effects meta-analysis and meta-regression of difference in mean haemoglobin were carried out, with subgroups defined by comparison of type of blood collection and analytic approach within each survey pair.ResultsWe included 23 survey pairs from 17 countries for PSC and 17 survey pairs from 11 countries for WRA. Meta-regression indicates that surveys measuring haemoglobin with HemoCue® Hb 301 found higher haemoglobin concentrations than near-in-time surveys using HemoCue® Hb 201+ in non-pregnant women ((NPW); 5.8 g/L (95% confidence interval (CI) = 3.2-8.3) mean difference, n = 5 pairs) and PSC (4.3 g/L (1.4-7.2), n = 6). Surveys collecting venous blood found higher haemoglobin concentrations than near-in-time surveys collecting capillary blood in PSC (3.8 g/L (0.8-6.7), n = 8), but not NPW (0.4 g/L (-1.9-2.8), n = 9).ConclusionsBecause this study is observational, differences in haemoglobin concentrations in near-in-time surveys may be caused by other factors associated with choice of analytic approach or type of blood collected. The source or sources of differences should be clarified to improve use of surveys to prioritize and evaluate public health programs.RegistrationPROSPERO CRD42022296553.

The development of next-generation antimalarials that are efficacious against the human liver and asexual blood stages is recognized as one of the worlds most pressing public health challenges. In recent years, aminoacyl-tRNA synthetases, including prolyl-tRNA synthetase, have emerged as attractive targets for malaria chemotherapy. We describe the development of a single-step biochemical assay for Plasmodium and human prolyl-tRNA synthetases that overcomes critical limitations of existing technologies and enables quantitative inhibitor profiling with high sensitivity and flexibility. Supported by this assay platform and co-crystal structures of representative inhibitor-target complexes, we develop a set of high-affinity prolyl-tRNA synthetase inhibitors, including previously elusive aminoacyl-tRNA synthetase triple-site ligands that simultaneously engage all three substrate-binding pockets. Several compounds exhibit potent dual-stage activity against Plasmodium parasites and display good cellular host selectivity. Our data inform the inhibitor requirements to overcome existing resistance mechanisms and establish a path for rational development of prolyl-tRNA synthetase-targeted anti-malarial therapies.

OBJECTIVE: The aim of the study was to determine whether tibiofemoral contact stress predicts risk for worsening knee pain over 84 ms in adults aged 50-79 yrs with or at elevated risk for knee osteoarthritis. DESIGN: Baseline tibiofemoral contact stress was estimated using discrete element analysis. Other baseline measures included weight, height, hip-knee-ankle alignment, Kellgren-Lawrence grade, and Western Ontario and McMaster Universities Osteoarthritis Index pain subscale. Logistic regression models assessed the association between baseline contact stress and 84-mo worsening of Western Ontario and McMaster Universities Osteoarthritis Index pain subscale. RESULTS: Data from the dominant knee (72.6% Kellgren-Lawrence grade 0/1 and 27.4% Kellgren-Lawrence grade ≥ 2) of 208 participants (64.4% female, mean ± SD body mass index = 29.6 ± 5.1 kg/m 2 ) were analyzed. Baseline mean and peak contact stress were 3.3 ± 0.9 and 9.4 ± 4.3 MPa, respectively. Forty-seven knees met the criterion for worsening pain. The highest tertiles in comparison with the lowest tertiles of mean (odds ratio [95% confidence interval] = 2.47 [1.03-5.95], P = 0.04) and peak (2.49 [1.03-5.98], P = 0.04) contact stress were associated with worsening pain at 84 mos, after adjustment for age, sex, race, clinic site, and baseline pain. Post hoc sensitivity analyses including adjustment for body mass index and hip-knee-ankle alignment attenuated the effect. CONCLUSIONS: These findings suggest that elevated tibiofemoral contact stress can predict the development of worsening of knee pain.

Aberrant transcriptional activity of androgen receptor (AR) is one of the dominant mechanisms for developing of castration-resistant prostate cancer (CRPC). Analyzing AR-transcriptional complex related to CRPC is therefore important towards understanding the mechanism of therapy resistance. While studying its mechanism, we observed that a transmembrane protein called neuropilin-2 (NRP2) plays a contributory role in forming a novel AR-transcriptional complex containing nuclear pore proteins. Using immunogold electron microscopy, high-resolution confocal microscopy, chromatin immunoprecipitation, proteomics, and other biochemical techniques, we delineated the molecular mechanism of how a specific splice variant of NRP2 becomes sumoylated upon ligand stimulation and translocates to the inner nuclear membrane. This splice variant of NRP2 then stabilizes the complex between AR and nuclear pore proteins to promote CRPC specific gene expression. Both full-length and splice variants of AR have been identified in this specific transcriptional complex. In vitro cell line-based assays indicated that depletion of NRP2 not only destabilizes the AR-nuclear pore protein interaction but also inhibits the transcriptional activities of AR. Using an in vivo bone metastasis model, we showed that the inhibition of NRP2 led to the sensitization of CRPC cells toward established anti-AR therapies such as enzalutamide. Overall, our finding emphasize the importance of combinatorial inhibition of NRP2 and AR as an effective therapeutic strategy against treatment refractory prostate cancer.

Prophylactic vaccines capable of preventing human papillomavirus (HPV) infections are still inaccessible to a vast majority of the global population due to their high cost and challenges related to multiple administrations performed in a medical setting. In an effort to improve distribution and administration, we have developed dissolvable microneedles loaded with a thermally stable HPV vaccine candidate consisting of Qβ virus-like particles (VLPs) displaying a highly conserved epitope from the L2 protein of HPV (Qβ-HPV). Polymeric microneedle delivery of Qβ-HPV produces similar amounts of anti-HPV16 L2 IgG antibodies compared to traditional subcutaneous injection while delivering a much smaller amount of intradermal dose. However, a dose sparing effect was found. Furthermore, immunization yielded neutralizing antibody responses in a HPV pseudovirus assay. The vaccine candidate was confirmed to be stable at room temperature after storage for several months, potentially mitigating many of the challenges associated with cold-chain distribution. The ease of self-administration and minimal invasiveness of such microneedle patch vaccines may enable wide-scale distribution of the HPV vaccine and lead to higher patient compliance. The Qβ VLP and its delivery technology is a plug-and-play system that could serve as a universal platform with a broad range of applications. Qβ VLPs may be stockpiled for conjugation to a wide range of epitopes, which are then packaged and delivered directly to the patient via noninvasive microneedle patches. Such a system paves the way for rapid distribution and self-administration of vaccines.

We identify the Plasmodium falciparum acetyl-coenzyme A synthetase (PfAcAS) as a druggable target, using genetic and chemical validation. In vitro evolution of resistance with two antiplasmodial drug-like compounds (MMV019721 and MMV084978) selects for mutations in PfAcAS. Metabolic profiling of compound-treated parasites reveals changes in acetyl-CoA levels for both compounds. Genome editing confirms that mutations in PfAcAS are sufficient to confer resistance. Knockdown studies demonstrate that PfAcAS is essential for asexual growth, and partial knockdown induces hypersensitivity to both compounds. In vitro biochemical assays using recombinantly expressed PfAcAS validates that MMV019721 and MMV084978 directly inhibit the enzyme by preventing CoA and acetate binding, respectively. Immunolocalization studies reveal that PfAcAS is primarily localized to the nucleus. Functional studies demonstrate inhibition of histone acetylation in compound-treated wild-type, but not in resistant parasites. Our findings identify and validate PfAcAS as an essential, druggable target involved in the epigenetic regulation of gene expression.

There is a shift in antimalarial drug discovery from phenotypic screening toward target-based approaches, as more potential drug targets are being validated in Plasmodium species. Given the high attrition rate and high cost of drug discovery, it is important to select the targets most likely to deliver progressible drug candidates. In this paper, we describe the criteria that we consider important for selecting targets for antimalarial drug discovery. We describe the analysis of a number of drug targets in the Malaria Drug Accelerator (MalDA) pipeline, which has allowed us to prioritize targets that are ready to enter the drug discovery process. This selection process has also highlighted where additional data are required to inform target progression or deprioritization of other targets. Finally, we comment on how additional drug targets may be identified.

Throughout development and aging, human cells accumulate mutations resulting in genomic mosaicism and genetic diversity at the cellular level. Mosaic mutations present in the gonads can affect both the individual and the offspring and subsequent generations. Here, we explore patterns and temporal stability of clonal mosaic mutations in male gonads by sequencing ejaculated sperm. Through 300× whole-genome sequencing of blood and sperm from healthy men, we find each ejaculate carries on average 33.3 ± 12.1 (mean ± SD) clonal mosaic variants, nearly all of which are detected in serial sampling, with the majority absent from sampled somal tissues. Their temporal stability and mutational signature suggest origins during embryonic development from a largely immutable stem cell niche. Clonal mosaicism likely contributes a transmissible, predicted pathogenic exonic variant for 1 in 15 men, representing a life-long threat of transmission for these individuals and a significant burden on human population health.

Malaria and iron deficiency (ID) are common and interrelated public health problems in African children. Observational data suggest that interrupting malaria transmission reduces the prevalence of ID1. To test the hypothesis that malaria might cause ID, we used sickle cell trait (HbAS, rs334 ), a genetic variant that confers specific protection against malaria2, as an instrumental variable in Mendelian randomization analyses. HbAS was associated with a 30% reduction in ID among children living in malaria-endemic countries in Africa (n = 7,453), but not among individuals living in malaria-free areas (n = 3,818). Genetically predicted malaria risk was associated with an odds ratio of 2.65 for ID per unit increase in the log incidence rate of malaria. This suggests that an intervention that halves the risk of malaria episodes would reduce the prevalence of ID in African children by 49%.

BACKGROUND: RET mutations occur in 70% of medullary thyroid cancers, and RET fusions occur rarely in other thyroid cancers. In patients with RET-altered thyroid cancers, the efficacy and safety of selective RET inhibition are unknown. METHODS: We enrolled patients with RET-mutant medullary thyroid cancer with or without previous vandetanib or cabozantinib treatment, as well as those with previously treated RET fusion-positive thyroid cancer, in a phase 1-2 trial of selpercatinib. The primary end point was an objective response (a complete or partial response), as determined by an independent review committee. Secondary end points included the duration of response, progression-free survival, and safety. RESULTS: In the first 55 consecutively enrolled patients with RET-mutant medullary thyroid cancer who had previously received vandetanib, cabozantinib, or both, the percentage who had a response was 69% (95% confidence interval [CI], 55 to 81), and 1-year progression-free survival was 82% (95% CI, 69 to 90). In 88 patients with RET-mutant medullary thyroid cancer who had not previously received vandetanib or cabozantinib, the percentage who had a response was 73% (95% CI, 62 to 82), and 1-year progression-free survival was 92% (95% CI, 82 to 97). In 19 patients with previously treated RET fusion-positive thyroid cancer, the percentage who had a response was 79% (95% CI, 54 to 94), and 1-year progression-free survival was 64% (95% CI, 37 to 82). The most common adverse events of grade 3 or higher were hypertension (in 21% of the patients), increased alanine aminotransferase level (in 11%), increased aspartate aminotransferase level (in 9%), hyponatremia (in 8%), and diarrhea (in 6%). Of all 531 patients treated, 12 (2%) discontinued selpercatinib owing to drug-related adverse events. CONCLUSIONS: In this phase 1-2 trial, selpercatinib showed durable efficacy with mainly low-grade toxic effects in patients with medullary thyroid cancer with and without previous vandetanib or cabozantinib treatment. (Funded by Loxo Oncology and others; LIBRETTO-001 ClinicalTrials.gov number, NCT03157128.).

Currently, the only approved hepatitis C virus (HCV) treatment for children aged

De novo mutations arising on the paternal chromosome make the largest known contribution to autism risk, and correlate with paternal age at the time of conception. The recurrence risk for autism spectrum disorders is substantial, leading many families to decline future pregnancies, but the potential impact of assessing parental gonadal mosaicism has not been considered. We measured sperm mosaicism using deep-whole-genome sequencing, for variants both present in an offspring and evident only in father's sperm, and identified single-nucleotide, structural and short tandem-repeat variants. We found that mosaicism quantification can stratify autism spectrum disorders recurrence risk due to de novo mutations into a vast majority with near 0% recurrence and a small fraction with a substantially higher and quantifiable risk, and we identify novel mosaic variants at risk for transmission to a future offspring. This suggests, therefore, that genetic counseling would benefit from the addition of sperm mosaicism assessment.

De novo mutations arising on the paternal chromosome make the largest known contribution to autism risk, and correlate with paternal age at the time of conception. The recurrence risk for autism spectrum disorders is substantial, leading many families to decline future pregnancies, but the potential impact of assessing parental gonadal mosaicism has not been considered. We measured sperm mosaicism using deep-whole-genome sequencing, for variants both present in an offspring and evident only in father's sperm, and identified single-nucleotide, structural and short tandem-repeat variants. We found that mosaicism quantification can stratify autism spectrum disorders recurrence risk due to de novo mutations into a vast majority with near 0% recurrence and a small fraction with a substantially higher and quantifiable risk, and we identify novel mosaic variants at risk for transmission to a future offspring. This suggests, therefore, that genetic counseling would benefit from the addition of sperm mosaicism assessment.

To discover leads for next-generation chemoprotective antimalarial drugs, we tested more than 500,000 compounds for their ability to inhibit liver-stage development of luciferase-expressing Plasmodium spp. parasites (681 compounds showed a half-maximal inhibitory concentration of less than 1 micromolar). Cluster analysis identified potent and previously unreported scaffold families as well as other series previously associated with chemoprophylaxis. Further testing through multiple phenotypic assays that predict stage-specific and multispecies antimalarial activity distinguished compound classes that are likely to provide symptomatic relief by reducing asexual blood-stage parasitemia from those which are likely to only prevent malaria. Target identification by using functional assays, in vitro evolution, or metabolic profiling revealed 58 mitochondrial inhibitors but also many chemotypes possibly with previously unidentified mechanisms of action.

To discover leads for next-generation chemoprotective antimalarial drugs, we tested more than 500,000 compounds for their ability to inhibit liver-stage development of luciferase-expressing Plasmodium spp. parasites (681 compounds showed a half-maximal inhibitory concentration of less than 1 micromolar). Cluster analysis identified potent and previously unreported scaffold families as well as other series previously associated with chemoprophylaxis. Further testing through multiple phenotypic assays that predict stage-specific and multispecies antimalarial activity distinguished compound classes that are likely to provide symptomatic relief by reducing asexual blood-stage parasitemia from those which are likely to only prevent malaria. Target identification by using functional assays, in vitro evolution, or metabolic profiling revealed 58 mitochondrial inhibitors but also many chemotypes possibly with previously unidentified mechanisms of action.

The dynamic shape of the endoplasmic reticulum (ER) is a reflection of its wide variety of critical cell biological functions. Consequently, perturbation of ER-shaping proteins can cause a range of human phenotypes. Here, we describe three affected children (from two consanguineous families) who carry homozygous loss-of-function mutations in LNPK (previously known as KIAA1715); this gene encodes lunapark, which is proposed to serve as a curvature-stabilizing protein within tubular three-way junctions of the ER. All individuals presented with severe psychomotor delay, intellectual disability, hypotonia, epilepsy, and corpus callosum hypoplasia, and two of three showed mild cerebellar hypoplasia and atrophy. Consistent with a proposed role in neurodevelopmental disease, LNPK was expressed during brain development in humans and mice and was present in neurite-like processes in differentiating human neural progenitor cells. Affected cells showed the absence of full-length lunapark, aberrant ER structures, and increased luminal mass density. Together, our results implicate the ER junction stabilizer lunapark in establishing the corpus callosum.

Study designThis is a focused review article.ObjectivesThis review presents important features of clinical outcomes assessments (COAs) in human spinal cord injury research. Considerations for COAs by trial phase and International Classification of Functioning, Disability and Health are presented as well as strengths and recommendations for upper extremity COAs for research. Clinical trial tools and designs to address recruitment challenges are identified.MethodsThe methods include a summary of topics discussed during a two-day workshop, conceptual discussion of upper extremity COAs and additional focused literature review.ResultsCOAs must be appropriate to trial phase and particularly in mid-late-phase trials, should reflect recovery vs. compensation, as well as being clinically meaningful. The impact and extent of upper vs. lower motoneuron disease should be considered, as this may affect how an individual may respond to a given therapeutic. For trials with broad inclusion criteria, the content of COAs should cover all severities and levels of SCI. Specific measures to assess upper extremity function as well as more comprehensive COAs are under development. In addition to appropriate use of COAs, methods to increase recruitment, such as adaptive trial designs and prognostic modeling to prospectively stratify heterogeneous populations into appropriate cohorts should be considered.ConclusionsWith an increasing number of clinical trials focusing on improving upper extremity function, it is essential to consider a range of factors when choosing a COA.SponsorsCraig H. Neilsen Foundation, Spinal Cord Outcomes Partnership Endeavor.

Study designThis is a focused review article.ObjectivesThis review presents important features of clinical outcomes assessments (COAs) in human spinal cord injury research. Considerations for COAs by trial phase and International Classification of Functioning, Disability and Health are presented as well as strengths and recommendations for upper extremity COAs for research. Clinical trial tools and designs to address recruitment challenges are identified.MethodsThe methods include a summary of topics discussed during a two-day workshop, conceptual discussion of upper extremity COAs and additional focused literature review.ResultsCOAs must be appropriate to trial phase and particularly in mid-late-phase trials, should reflect recovery vs. compensation, as well as being clinically meaningful. The impact and extent of upper vs. lower motoneuron disease should be considered, as this may affect how an individual may respond to a given therapeutic. For trials with broad inclusion criteria, the content of COAs should cover all severities and levels of SCI. Specific measures to assess upper extremity function as well as more comprehensive COAs are under development. In addition to appropriate use of COAs, methods to increase recruitment, such as adaptive trial designs and prognostic modeling to prospectively stratify heterogeneous populations into appropriate cohorts should be considered.ConclusionsWith an increasing number of clinical trials focusing on improving upper extremity function, it is essential to consider a range of factors when choosing a COA.SponsorsCraig H. Neilsen Foundation, Spinal Cord Outcomes Partnership Endeavor.

DesignUniversal voluntary HIV counselling and testing followed by prompt initiation of antiretroviral therapy (ART) for all those diagnosed HIV-infected (universal test and treat, UTT) is now a global health standard. However, its population-level impact, feasibility and cost remain unknown. Five community-based trials have been implemented in sub-Saharan Africa to measure the effects of various UTT strategies at population level: BCPP/YaTsie in Botswana, MaxART in Swaziland, HPTN 071 (PopART) in South Africa and Zambia, SEARCH in Uganda and Kenya and ANRS 12249 TasP in South Africa. This report describes and contrasts the contexts, research methodologies, intervention packages, themes explored, evolution of study designs and interventions related to each of these five UTT trials.MethodsWe conducted a comparative assessment of the five trials using data extracted from study protocols and collected during baseline studies, with additional input from study investigators. We organized differences and commonalities across the trials in five categories: trial contexts, research designs, intervention packages, trial themes and adaptations.ResultsAll performed in the context of generalized HIV epidemics, the trials highly differ in their social, demographic, economic, political and health systems settings. They share the common aim of assessing the impact of UTT on the HIV epidemic but differ in methodological aspects such as study design and eligibility criteria for trial populations. In addition to universal ART initiation, the trials deliver a wide range of biomedical, behavioural and structural interventions as part of their UTT strategies. The five studies explore common issues, including the uptake rates of the trial services and individual health outcomes. All trials have adapted since their initiation to the evolving political, economic and public health contexts, including adopting the successive national recommendations for ART initiation.ConclusionsWe found substantial commonalities but also differences between the five UTT trials in their design, conduct and multidisciplinary outputs. As empirical literature on how UTT may improve efficiency and quality of HIV care at population level is still scarce, this article provides a foundation for more collaborative research on UTT and supports evidence-based decision making for HIV care in country and internationally.

DesignUniversal voluntary HIV counselling and testing followed by prompt initiation of antiretroviral therapy (ART) for all those diagnosed HIV-infected (universal test and treat, UTT) is now a global health standard. However, its population-level impact, feasibility and cost remain unknown. Five community-based trials have been implemented in sub-Saharan Africa to measure the effects of various UTT strategies at population level: BCPP/YaTsie in Botswana, MaxART in Swaziland, HPTN 071 (PopART) in South Africa and Zambia, SEARCH in Uganda and Kenya and ANRS 12249 TasP in South Africa. This report describes and contrasts the contexts, research methodologies, intervention packages, themes explored, evolution of study designs and interventions related to each of these five UTT trials.MethodsWe conducted a comparative assessment of the five trials using data extracted from study protocols and collected during baseline studies, with additional input from study investigators. We organized differences and commonalities across the trials in five categories: trial contexts, research designs, intervention packages, trial themes and adaptations.ResultsAll performed in the context of generalized HIV epidemics, the trials highly differ in their social, demographic, economic, political and health systems settings. They share the common aim of assessing the impact of UTT on the HIV epidemic but differ in methodological aspects such as study design and eligibility criteria for trial populations. In addition to universal ART initiation, the trials deliver a wide range of biomedical, behavioural and structural interventions as part of their UTT strategies. The five studies explore common issues, including the uptake rates of the trial services and individual health outcomes. All trials have adapted since their initiation to the evolving political, economic and public health contexts, including adopting the successive national recommendations for ART initiation.ConclusionsWe found substantial commonalities but also differences between the five UTT trials in their design, conduct and multidisciplinary outputs. As empirical literature on how UTT may improve efficiency and quality of HIV care at population level is still scarce, this article provides a foundation for more collaborative research on UTT and supports evidence-based decision making for HIV care in country and internationally.

BackgroundPrimary analysis of the double-blind, phase III Efficacy of XL184 (Cabozantinib) in Advanced Medullary Thyroid Cancer (EXAM) trial demonstrated significant improvement in progression-free survival with cabozantinib versus placebo in patients with progressive medullary thyroid cancer (MTC). Final analysis of overall survival (OS), a key secondary endpoint, was carried out after long-term follow-up.Patients and methodsEXAM compared cabozantinib with placebo in 330 patients with documented radiographic progression of metastatic MTC. Patients were randomized (2:1) to cabozantinib (140 mg/day) or placebo. Final OS and updated safety data are reported.ResultsMinimum follow-up was 42 months. Kaplan-Meier analysis showed a 5.5-month increase in median OS with cabozantinib versus placebo (26.6 versus 21.1 months) although the difference did not reach statistical significance [stratified hazard ratio (HR), 0.85; 95% confidence interval (CI), 0.64-1.12; P = 0.24]. In an exploratory assessment of OS, progression-free survival, and objective response rate, cabozantinib appeared to have a larger treatment effect in patients with RET M918T mutation-positive tumors compared with patients not harboring this mutation. For patients with RET M918T-positive disease, median OS was 44.3 months for cabozantinib versus 18.9 months for placebo [HR, 0.60; 95% CI, 0.38-0.94; P = 0.03 (not adjusted for multiple subgroup analyses)], with corresponding values of 20.2 versus 21.5 months (HR, 1.12; 95% CI, 0.70-1.82; P = 0.63) in the RET M918T-negative subgroup. Median treatment duration was 10.8 months with cabozantinib and 3.4 months with placebo. The safety profile for cabozantinib remained consistent with that of the primary analysis.ConclusionThe secondary end point was not met in this final OS analysis from the trial of cabozantinib in patients with metastatic, radiographically progressive MTC. A statistically nonsignificant increase in OS was observed for cabozantinib compared with placebo. Exploratory analyses suggest that patients with RET M918T-positive tumors may experience a greater treatment benefit with cabozantinib.Trial registration numberNCT00704730.

BackgroundPrimary analysis of the double-blind, phase III Efficacy of XL184 (Cabozantinib) in Advanced Medullary Thyroid Cancer (EXAM) trial demonstrated significant improvement in progression-free survival with cabozantinib versus placebo in patients with progressive medullary thyroid cancer (MTC). Final analysis of overall survival (OS), a key secondary endpoint, was carried out after long-term follow-up.Patients and methodsEXAM compared cabozantinib with placebo in 330 patients with documented radiographic progression of metastatic MTC. Patients were randomized (2:1) to cabozantinib (140 mg/day) or placebo. Final OS and updated safety data are reported.ResultsMinimum follow-up was 42 months. Kaplan-Meier analysis showed a 5.5-month increase in median OS with cabozantinib versus placebo (26.6 versus 21.1 months) although the difference did not reach statistical significance [stratified hazard ratio (HR), 0.85; 95% confidence interval (CI), 0.64-1.12; P = 0.24]. In an exploratory assessment of OS, progression-free survival, and objective response rate, cabozantinib appeared to have a larger treatment effect in patients with RET M918T mutation-positive tumors compared with patients not harboring this mutation. For patients with RET M918T-positive disease, median OS was 44.3 months for cabozantinib versus 18.9 months for placebo [HR, 0.60; 95% CI, 0.38-0.94; P = 0.03 (not adjusted for multiple subgroup analyses)], with corresponding values of 20.2 versus 21.5 months (HR, 1.12; 95% CI, 0.70-1.82; P = 0.63) in the RET M918T-negative subgroup. Median treatment duration was 10.8 months with cabozantinib and 3.4 months with placebo. The safety profile for cabozantinib remained consistent with that of the primary analysis.ConclusionThe secondary end point was not met in this final OS analysis from the trial of cabozantinib in patients with metastatic, radiographically progressive MTC. A statistically nonsignificant increase in OS was observed for cabozantinib compared with placebo. Exploratory analyses suggest that patients with RET M918T-positive tumors may experience a greater treatment benefit with cabozantinib.Trial registration numberNCT00704730.

Pontocerebellar hypoplasia (PCH) represents a group of recessive developmental disorders characterized by impaired growth of the pons and cerebellum, which frequently follows a degenerative course. Currently, there are 10 partially overlapping clinical subtypes and 13 genes known mutated in PCH. Here, we report biallelic TBC1D23 mutations in six individuals from four unrelated families manifesting a non-degenerative form of PCH. In addition to reduced volume of pons and cerebellum, affected individuals had microcephaly, psychomotor delay, and ataxia. In zebrafish, tbc1d23 morphants replicated the human phenotype showing hindbrain volume loss. TBC1D23 localized at the trans-Golgi and was regulated by the small GTPases Arl1 and Arl8, suggesting a role in trans-Golgi membrane trafficking. Altogether, this study provides a causative link between TBC1D23 mutations and PCH and suggests a less severe clinical course than other PCH subtypes.

Since its introduction 20 years ago for the treatment of chronic wounds, negative pressure wound therapy use has expanded to a variety of other wound types. Various mechanisms of action for its efficacy in wound healing have been postulated, but no unifying theory exists. Proposed mechanisms include induction of perfusion changes, microdeformation, macrodeformation, exudate control and decreasing the bacterial load in the wound. We surmise that these different mechanisms have varying levels of dominance in each wound type. Specifically, negative pressure wound therapy is beneficial to acute open wounds because it induces perfusion changes and formation of granulation tissue. Post-surgical incisional wounds are positively affected by perfusion changes and exudate control. In the context of chronic wounds, negative pressure wound therapy removes harmful and corrosive substances within the wounds to affect healing. When skin grafts and dermal substitutes are used to close a wound, negative pressure wound therapy is effective in promoting granulation tissue formation, controlling exudate and decreasing the bacterial load in the wound. In this review, we elucidate some of the mechanisms behind the positive wound healing effects of negative pressure wound therapy, providing possible explanations for these effects in different wound types.

Background: Anemia in women of reproductive age (WRA) (age range: 15-49 y) remains a public health problem globally, and reducing anemia in women by 50% by 2025 is a goal of the World Health Assembly.Objective: We assessed the associations between anemia and multiple proximal risk factors (e.g., iron and vitamin A deficiencies, inflammation, malaria, and body mass index) and distal risk factors (e.g., education status, household sanitation and hygiene, and urban or rural residence) in nonpregnant WRA.Design: Cross-sectional, nationally representative data from 10 surveys (n = 27,018) from the Biomarkers Reflecting Inflammation and Nutritional Determinants of Anemia (BRINDA) project were analyzed individually and pooled by the infection burden and risk in the country. We examined the severity of anemia and measured the bivariate associations between anemia and factors at the country level and by infection burden, which we classified with the use of the national prevalences of malaria, HIV, schistosomiasis, sanitation, and water-quality indicators. Pooled multivariate logistic regression models were constructed for each infection-burden category to identify independent determinants of anemia (hemoglobin concertation

Background: The accurate estimation of the prevalence of vitamin A deficiency (VAD) is important in planning and implementing interventions. Retinol-binding protein (RBP) is often used in population surveys to measure vitamin A status, but its interpretation is challenging in settings where inflammation is common because RBP concentrations decrease during the acute-phase response.Objectives: We aimed to assess the relation between RBP concentrations and inflammation and malaria in preschool children (PSC) (age range: 6-59 mo) and women of reproductive age (WRA) (age range: 15-49 y) and to investigate adjustment algorithms to account for these effects.Design: Cross-sectional data from 8 surveys for PSC (n = 8803) and 4 surveys for WRA (n = 4191) from the Biomarkers Reflecting Inflammation and Nutritional Determinants of Anemia (BRINDA) project were analyzed individually and combined with the use of a meta-analysis. Several approaches were explored to adjust RBP concentrations in PSC in inflammation and malaria settings as follows: 1) the exclusion of subjects with C-reactive protein (CRP) concentrations >5 mg/L or α-1-acid glycoprotein (AGP) concentrations >1 g/L, 2) the application of arithmetic correction factors, and 3) the use of a regression correction approach. The impact of adjustment on the estimated prevalence of VAD (defined as

Background: A lack of information on the etiology of anemia has hampered the design and monitoring of anemia-control efforts.Objective: We aimed to evaluate predictors of anemia in preschool children (PSC) (age range: 6-59 mo) by country and infection-burden category.Design: Cross-sectional data from 16 surveys (n = 29,293) from the Biomarkers Reflecting Inflammation and Nutritional Determinants of Anemia (BRINDA) project were analyzed separately and pooled by category of infection burden. We assessed relations between anemia (hemoglobin concentration 50% of surveys. Associations between breastfeeding and anemia were attenuated by controlling for child age, which was negatively associated with anemia. The most consistent predictors of severe anemia were malaria, poor sanitation, and underweight. In multivariable pooled models, child age, iron deficiency, and stunting independently predicted anemia and severe anemia. Inflammation was generally associated with anemia in the high- and very high-infection groups but not in the low- and medium-infection groups. In PSC with anemia, 50%, 30%, 55%, and 58% of children had concomitant iron deficiency in low-, medium-, high-, and very high-infection categories, respectively.Conclusions: Although causal inference is limited by cross-sectional survey data, results suggest anemia-control programs should address both iron deficiency and infections. The relative importance of factors that are associated with anemia varies by setting, and thus, country-specific data are needed to guide programs.

Background: A lack of information on the etiology of anemia has hampered the design and monitoring of anemia-control efforts.Objective: We aimed to evaluate predictors of anemia in preschool children (PSC) (age range: 6-59 mo) by country and infection-burden category.Design: Cross-sectional data from 16 surveys (n = 29,293) from the Biomarkers Reflecting Inflammation and Nutritional Determinants of Anemia (BRINDA) project were analyzed separately and pooled by category of infection burden. We assessed relations between anemia (hemoglobin concentration 50% of surveys. Associations between breastfeeding and anemia were attenuated by controlling for child age, which was negatively associated with anemia. The most consistent predictors of severe anemia were malaria, poor sanitation, and underweight. In multivariable pooled models, child age, iron deficiency, and stunting independently predicted anemia and severe anemia. Inflammation was generally associated with anemia in the high- and very high-infection groups but not in the low- and medium-infection groups. In PSC with anemia, 50%, 30%, 55%, and 58% of children had concomitant iron deficiency in low-, medium-, high-, and very high-infection categories, respectively.Conclusions: Although causal inference is limited by cross-sectional survey data, results suggest anemia-control programs should address both iron deficiency and infections. The relative importance of factors that are associated with anemia varies by setting, and thus, country-specific data are needed to guide programs.

Background: The accurate estimation of the prevalence of vitamin A deficiency (VAD) is important in planning and implementing interventions. Retinol-binding protein (RBP) is often used in population surveys to measure vitamin A status, but its interpretation is challenging in settings where inflammation is common because RBP concentrations decrease during the acute-phase response.Objectives: We aimed to assess the relation between RBP concentrations and inflammation and malaria in preschool children (PSC) (age range: 6-59 mo) and women of reproductive age (WRA) (age range: 15-49 y) and to investigate adjustment algorithms to account for these effects.Design: Cross-sectional data from 8 surveys for PSC (n = 8803) and 4 surveys for WRA (n = 4191) from the Biomarkers Reflecting Inflammation and Nutritional Determinants of Anemia (BRINDA) project were analyzed individually and combined with the use of a meta-analysis. Several approaches were explored to adjust RBP concentrations in PSC in inflammation and malaria settings as follows: 1) the exclusion of subjects with C-reactive protein (CRP) concentrations >5 mg/L or α-1-acid glycoprotein (AGP) concentrations >1 g/L, 2) the application of arithmetic correction factors, and 3) the use of a regression correction approach. The impact of adjustment on the estimated prevalence of VAD (defined as

Background: Anemia in women of reproductive age (WRA) (age range: 15-49 y) remains a public health problem globally, and reducing anemia in women by 50% by 2025 is a goal of the World Health Assembly.Objective: We assessed the associations between anemia and multiple proximal risk factors (e.g., iron and vitamin A deficiencies, inflammation, malaria, and body mass index) and distal risk factors (e.g., education status, household sanitation and hygiene, and urban or rural residence) in nonpregnant WRA.Design: Cross-sectional, nationally representative data from 10 surveys (n = 27,018) from the Biomarkers Reflecting Inflammation and Nutritional Determinants of Anemia (BRINDA) project were analyzed individually and pooled by the infection burden and risk in the country. We examined the severity of anemia and measured the bivariate associations between anemia and factors at the country level and by infection burden, which we classified with the use of the national prevalences of malaria, HIV, schistosomiasis, sanitation, and water-quality indicators. Pooled multivariate logistic regression models were constructed for each infection-burden category to identify independent determinants of anemia (hemoglobin concertation

Purpose There are no approved treatments for recurrent/metastatic head and neck squamous cell carcinoma refractory to platinum and cetuximab. In the single-arm, phase II KEYNOTE-055 study, we evaluated pembrolizumab, an anti-programmed death 1 receptor antibody, in this platinum- and cetuximab-pretreated population with poor prognosis. Methods Eligibility stipulated disease progression within 6 months of platinum and cetuximab treatment. Patients received pembrolizumab 200 mg every 3 weeks. Imaging was performed every 6 to 9 weeks. Primary end points: overall response rate (Response Evaluation Criteria in Solid Tumors v1.1, central review) and safety. Efficacy was assessed in all dosed patients and in subgroups on the basis of programmed death ligand 1 (PD-L1) expression and human papillomavirus (HPV) status. Results Among 171 patients treated, 75% received two or more prior lines of therapy for metastatic disease, 82% were PD-L1 positive, and 22% were HPV positive. At the time of analysis, 109 patients (64%) experienced a treatment-related adverse event; 26 patients (15%) experienced a grade ≥ 3 event. Seven patients (4%) discontinued treatment, and one died of treatment-related adverse events. Overall response rate was 16% (95% CI, 11% to 23%), with a median duration of response of 8 months (range, 2+ to 12+ months); 75% of responses were ongoing at the time of analysis. Response rates were similar in all HPV and PD-L1 subgroups. Median progression-free survival was 2.1 months, and median overall survival was 8 months. Conclusion Pembrolizumab exhibited clinically meaningful antitumor activity and an acceptable safety profile in recurrent/metastatic head and neck squamous cell carcinoma previously treated with platinum and cetuximab.

Current embodiments of negative pressure wound therapy (NPWT) create a hermetically sealed chamber at the surface of the body using polyurethane foam connected to a vacuum pump, which is then covered by a flexible adhesive drape. Commercially available NPWT systems routinely use flexible polyethylene films that have a sticky side, coated with the same acrylate adhesives used in other medical devices such as ECG leads and grounding pads. Severe reactions to the acrylate adhesives in these other devices, although uncommon, have been reported. We describe the case of a 63-year-old woman with an intractable leg ulcer resulting from external-beam radiotherapy (XRT). Treatment with a standard commercial NPWT system induced severe inflammation of the skin in direct contact with drape adhesive. We successfully administered prolonged, outpatient NPWT to the patient using an alternative method (first described by Bagautdinov in 1986), using plain polyethylene film and petrolatum. The necessary hermetic seal is achieved by smearing the skin with petrolatum before applying the polyethylene film and activating the vacuum pump. The Bagautdinov method is a practical solution to the problem of adapting NPWT to patients with contact sensitivity or skin tears related to the adhesive compounds in the flexible drapes. Its use of a circumferential elastic wrap to maintain constant pressure on the seal probably limits the Bagautdinov technique to the extremities.

BackgroundAbout half of patients with papillary thyroid cancer have tumours with activating BRAF(V600E) mutations. Vemurafenib, an oncogenic BRAF kinase inhibitor approved for BRAF-positive melanoma, showed clinical benefit in three patients with BRAF(V600E)-positive papillary thyroid cancer in a phase 1 trial. We aimed to establish the activity of vemurafenib in patients with BRAF(V600E)-positive papillary thyroid cancer.MethodsWe did an open-label, non-randomised, phase 2 trial at ten academic centres and hospitals worldwide in patients aged 18 years or older with histologically confirmed recurrent or metastatic papillary thyroid cancer refractory to radioactive iodine and positive for the BRAF(V600E) mutation. Participants either had never received a multikinase inhibitor targeting VEGFR (cohort 1) or had been treated previously with a VEGFR multikinase inhibitor (cohort 2). Patients received vemurafenib 960 mg orally twice daily. The primary endpoint was investigator-assessed best overall response in cohort 1 (confirmed on two assessments 4 weeks or longer apart). Analyses were planned to have a minimum median follow-up of 15 months (data cutoff April 18, 2014) and were done in safety, intention-to-treat, and per-protocol populations. This trial is closed and is registered at ClinicalTrials.gov, number NCT01286753.FindingsBetween June 23, 2011, and Jan 15, 2013, 51 patients were enrolled to the study, 26 in cohort 1 and 25 in cohort 2. Median duration of follow-up was 18·8 months (IQR 14·2-26·0) in cohort 1 and 12·0 months (6·7-20·3) in cohort 2. Partial responses were recorded in ten of 26 patients in cohort 1 (best overall response 38·5%, 95% CI 20·2-59·4). Grade 3 or 4 adverse events were recorded in 17 (65%) of 26 patients in cohort 1 and 17 (68%) of 25 patients in cohort 2; the most common grade 3 and 4 adverse events were squamous cell carcinoma of the skin (seven [27%] in cohort 1, five [20%] in cohort 2), lymphopenia (two [8%] in each cohort), and increased γ-glutamyltransferase (one [4%] in cohort 1, three [12%] in cohort 2). Two individuals in cohort 2 died due to adverse events, one from dyspnoea and one from multiorgan failure, but neither was treatment related. Serious adverse events were reported for 16 (62%) of 26 patients in cohort 1 and 17 (68%) of 25 patients in cohort 2.InterpretationVemurafenib showed antitumour activity in patients with progressive, BRAF(V600E)-positive papillary thyroid cancer refractory to radioactive iodine who had never been treated with a multikinase inhibitor. As such, this agent represents a potential new treatment option for these patients.FundingF Hoffmann-La Roche.

BackgroundOptimization of fat grafting continues to gain increasing attention in the field of regenerative medicine. "Nanofat grafting" implements mechanical emulsification and injection of standard lipoaspirate for the correction of superficial rhytides and skin discoloration; however, little is known about the cellular constituents of the graft. Based on recent evidence that various stressors can induce progenitor activity, the authors hypothesized that the shear forces used in common fat grafting techniques may impact their regenerative capacities.MethodsLipoaspirates were obtained from 10 patients undergoing elective procedures. Half of each sample was subjected to nanofat processing; the other half was left unchallenged. The viscosity of each sample was measured for computational analysis. The stromal vascular fraction of each sample was isolated, quantified, and analyzed by means of flow cytometry with two multicolor fluorescence antibody panels.ResultsStandard lipoaspirate is ideally suited for mechanical stress induction. The mechanical emulsification involved in nanofat processing did not affect cell number; however, viability was greatly reduced when compared with the stromal vascular fraction of standard lipoaspirate. Interestingly, nanofat processing resulted in stress-induced stromal vascular fraction with a higher proportion of endothelial progenitor cells, mesenchymal stem cells, and multilineage differentiating stress-enduring cells. Single-parameter analysis also revealed significant increases in CD34, CD13, CD73, and CD146 of the stress-induced stromal vascular fraction, markers associated with mesenchymal stem cell activity.ConclusionsMechanical processing used in techniques such as nanofat grafting induces the up-regulation of progenitor phenotypes consistent with multipotency and pluripotency. These data provide a first step in characterizing the potential regenerative benefits realized through stress induction in fat grafting.Clincal question/level of evidenceTherapeutic, V.

ObjectiveTo evaluate whether change in fixed-location measures of radiographic joint space width (JSW) and cartilage thickness by MRI predict knee replacement.MethodsKnees replaced between 36 and 60 months' follow-up in the Osteoarthritis Initiative were each matched with one control by age, sex and radiographic status. Radiographic JSW was determined from fixed flexion radiographs and subregional femorotibial cartilage thickness from 3 T MRI. Changes between the annual visit before replacement (T0) and 2 years before T0 (T-2) were compared using conditional logistic regression.ResultsOne hundred and nineteen knees from 102 participants (55.5 % women; age 64.2 ± 8.7 [mean ± SD] years) were studied. Fixed-location JSW change at 22.5 % from medial to lateral differed more between replaced and control knees (case-control [cc] OR = 1.57; 95 % CI: 1.23-2.01) than minimum medial JSW change (ccOR = 1.38; 95 % CI: 1.11-1.71). Medial femorotibial cartilage loss displayed discrimination similar to minimum JSW, and central tibial cartilage loss similar to fixed-location JSW. Location-independent thinning and thickening scores were elevated prior to knee replacement.ConclusionsDiscrimination of structural progression between knee pre-placement cases versus controls was stronger for fixed-location than minimum radiographic JSW. MRI displayed similar discrimination to radiography and suggested greater simultaneous cartilage thickening and loss prior to knee replacement.Key points• Fixed-location JSW predicts surgical knee replacement more strongly than minimum JSW. • MRI predicts knee replacement with similar accuracy to radiographic JSW. • MRI reveals greater cartilage thinning and thickening prior to knee replacement.

Scarring following burn injury and its accompanying aesthetic and functional sequelae still pose major challenges. Hypertrophic scarring (HTS) can greatly impact patients' quality of life related to appearance, pain, pruritus and even loss of function of the injured body region. The identification of molecular events occurring in the evolution of the burn scar has increased our knowledge; however, this information has not yet translated into effective treatment modalities. Although many of the pathophysiologic pathways that bring about exaggerated scarring have been identified, certain nuances in burn scar formation are starting to be recognized. These include the effects of neurogenic inflammation, mechanotransduction, and the unique interactions of burn wound fluid with fat tissue in the deeper dermal layers, all of which may influence scarring outcome. Tension on the healing scar, pruritus, and pain all induce signaling pathways that ultimately result in increased collagen formation and myofibroblast phenotypic changes. Exposure of the fat domes in the deep dermis is associated with increased HTS, possibly on the basis of altered interaction of adipose-derived stem cells and the deep burn exudate. These pathophysiologic patterns related to stem cell-cytokine interactions, mechanotransduction, and neurogenic inflammation can provide new avenues of exploration for possible therapeutic interventions.

Fat grafting is commonly employed by plastic and reconstructive surgeons to address contour abnormalities and soft-tissue defects; however, because retention rates and thus volume filling effects are unpredictable, there is a search for new and innovative approaches. Initial studies on the use of human decellularized adipose tissue extracellular matrix (hDAM) show promise for its use not only in tissue engineering, but also in fat grafting. In this review, we examine and analyze the literature for the preparation, characterization, and use of hDAM and its derivatives in tissue engineering and plastic surgery applications. All studies reviewed involve physical, chemical, and/or biological treatment stages for the preparation of hDAM; however a distinction should be made between detergent and nondetergent-based processing, the latter of which appears to preserve the native integrity of the hDAM while most-efficiently achieving complete decellularization. Methods of hDAM characterization vary among groups and included simple and immunohistochemical staining, biochemical assays, 3-dimensional (3D) imaging, and mechano-stress testing, all of which are necessary to achieve a comprehensive description of this novel tissue. Finally, we examine the various preclinical models utilized to optimize hDAM performance, which primarily include the addition of adipose-derived stem cells or cross-linking agents. Overall, hDAM appears to be a promising adjunct in fat-grafting applications or even possibly as a stand-alone soft-tissue filler with off-the-shelf potential for commercial applications.

RationalePlasma-detectable biomarkers that rapidly and accurately diagnose bacterial infections in children with suspected pneumonia could reduce the morbidity of respiratory disease and decrease the unnecessary use of antibiotic therapy.ObjectivesUsing 56 markers measured in a multiplexed immunoassay, we sought to identify proteins and protein combinations that could discriminate bacterial from viral or malarial diagnoses.MethodsWe selected 80 patients with clinically diagnosed pneumonia (as defined by the World Health Organization) who also met criteria for bacterial, viral, or malarial infection based on clinical, radiographic, and laboratory results. Ten healthy community control subjects were enrolled to assess marker reliability. Patients were subdivided into two sets: one for identifying potential markers and another for validating them.Measurements and main resultsThree proteins (haptoglobin, tumor necrosis factor receptor 2 or IL-10, and tissue inhibitor of metalloproteinases 1) were identified that, when combined through a classification tree signature, accurately classified patients into bacterial, malarial, and viral etiologies and misclassified only one patient with bacterial pneumonia from the validation set. The overall sensitivity and specificity of this signature for the bacterial diagnosis were 96 and 86%, respectively. Alternative combinations of markers with comparable accuracy were selected by support vector machine and regression models and included haptoglobin, IL-10, and creatine kinase-MB.ConclusionsCombinations of plasma proteins accurately identified children with a respiratory syndrome who were likely to have bacterial infections and who would benefit from antibiotic therapy. When used in conjunction with malaria diagnostic tests, they may improve diagnostic specificity and simplify treatment decisions for clinicians.

ObjectivesTo provide a comprehensive simultaneous relation of various semiquantitative knee OA MRI features as well as the presence of baseline radiographic osteoarthritis (OA) to quantitative longitudinal cartilage loss.MethodsWe studied Multicenter OA Study (MOST) participants from a longitudinal observational study that included quantitative MRI measurement of cartilage thickness. These subjects also had Whole Organ MRI Score (WORMS) scoring of cartilage damage, bone marrow lesions (BMLs), meniscal pathology, and synovitis, as well as baseline radiographic evaluation for Kellgren and Lawrence (KL) grading. Knee compartments were classified as progressors when exceeding thresholds of measurement variability in normal knees. All potential risk factors of cartilage loss were dichotomized into "present" (score ≥2 for cartilage, ≥1 for others) or "absent". Differences in baseline scores of ipsi-compartmental risk factors were compared between progressor and non-progressor knees by multivariable logistic regression, adjusting for age, sex, body mass index, alignment axis (degrees) and baseline KL grade. Odds ratios (OR) and 95% CIs were calculated for medial femorotibial compartment (MFTC) and lateral femorotibial compartment (LFTC) cartilage loss. Cartilage loss across both compartments was studied using Generalized Estimating Equations.Results196 knees of 196 participants were included (age 59.8 ± 6.3 years [mean ± SD], BMI 29.5 ± 4.6, 62% women). For combined analyses of MFTC and LFTC, baseline factors related to cartilage loss were radiographic OA (KL grade ≥2: aOR 4.8 [2.4-9.5], cartilage damage (aOR 2.3 [1.2-4.4])), meniscal damage (aOR 3.9 [2.1-7.4]) and extrusion (aOR 2.9 [1.6-5.3]), all in the ipsilateral compartment, but not BMLs or synovitis.ConclusionBaseline radiographic OA and semiquantitatively (SQ) assessed MRI-detected cartilage damage, meniscal damage and extrusion, but not BMLs or synovitis is related to quantitatively measured ipsi-compartmental cartilage thinning over 30 months.

OBJECTIVE:To investigate the association of cartilage thickness change over 24 months, as determined by magnetic resonance imaging (MRI), with knee osteoarthritis (OA) progression at 24-48 months. METHODS:This nested case-control study included 600 knees with a baseline Kellgren/Lawrence (K/L) grade of 1-3 from 600 Osteoarthritis Initiative (OAI) participants. Case knees (n = 194) had both medial tibiofemoral radiographic joint space loss (≥0.7 mm) and a persistent increase in the Western Ontario and McMaster Universities Osteoarthritis Index pain score (≥9 on a 0-100 scale) 24-48 months from baseline. Control knees (n = 406) included 200 with neither radiographic nor pain progression, 103 with radiographic progression only, and 103 with pain progression only. Medial and lateral femorotibial cartilage was segmented from sagittal 3T MRIs at baseline, 12 months, and 24 months. Logistic regression was used to assess the association of change in cartilage thickness, with a focus on the central medial femorotibial compartment, and OA progression. RESULTS:Central medial femorotibial compartment thickness loss was significantly associated with case status, with an odds ratio (OR) of 1.9 (95% confidence interval [95% CI] 1.6-2.3) (P < 0.0001). Association with case status reached P < 0.05 for both the central femur (OR 1.8 [95% CI 1.5-2.2]) and the central tibia (OR 1.6 [95% CI 1.3-1.9]). Lateral femorotibial compartment cartilage thickness loss, in contrast, was not significantly associated with case status. A reduction in central medial femorotibial compartment cartilage thickness was strongly associated with radiographic progression (OR 4.0 [95% CI 2.9-5.3]; P < 0.0001) and only weakly associated with pain progression (OR 1.3 [95% CI 1.1-1.6]; P < 0.01). CONCLUSION:Our findings indicate that loss of medial femorotibial cartilage thickness over 24 months is associated with the combination of radiographic and pain progression in the knee, with a stronger association for radiographic progression.

The Ah receptor (AhR)-responsive CALUX (chemically activated luciferase expression) cell bioassay is commonly used for rapid screening of samples for the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin), dioxin-like compounds, and AhR agonists/antagonists. By increasing the number of AhR DNA recognition sites (dioxin responsive elements), we previously generated a novel third generation (G3) recombinant AhR-responsive mouse CALUX cell line (H1L7.5c3) with a significantly enhanced response to DLCs compared to existing AhR-CALUX cell bioassays. However, the elevated background luciferase activity of these cells and the absence of comparable G3 cell lines derived from other species have limited their utility for screening purposes. Here, we describe the development and characterization of species-specific G3 recombinant AhR-responsive CALUX cell lines (rat, human, and guinea pig) that exhibit significantly improved limit of detection and dramatically increased TCDD induction response. The low background luciferase activity, low minimal detection limit (0.1 pM TCDD) and enhanced induction response of the rat G3 cell line (H4L7.5c2) over the H1L7.5c3 mouse G3 cells, identifies them as a more optimal cell line for screening purposes. The utility of the new G3 CALUX cell lines were demonstrated by screening sediment extracts and a small chemical compound library for the presence of AhR agonists. The improved limit of detection and increased response of these new G3 CALUX cell lines will facilitate species-specific analysis of DLCs and AhR agonists in samples with low levels of contamination and/or in small sample volumes.

ObjectiveTo explore whether baseline to 12-month followup change in femorotibial cartilage thickness differs between subjects who received a total knee arthroplasty (TKA) between 24 and 60 months from those without TKA (non-TKA).MethodsIn this prospective cohort study, 531 right knees from Osteoarthritis Initiative participants with definite radiographic knee osteoarthritis (Kellgren/Lawrence [K/L] grades 2-4) were studied. Segmentation was applied to coronal fast low-angle shot magnetic resonance images, to quantitatively determine cartilage thickness in 16 femorotibial subregions. Unadjusted P values (t-tests) and P values adjusted for age, baseline body mass index (BMI), K/L grade, and sex (generalized estimating equation models) were used to evaluate differences in longitudinal 1-year rates of cartilage thickness between TKAs and non-TKAs, with total knee arthroplasty status as fixed effect.ResultsOf the 531 participants (mean ± SD ages 63 ± 9 years, BMI 30 ± 4.8 kg/m(2)), 40 received a femorotibial TKA within 4 years. At baseline, TKAs had thinner medial and lateral femorotibial cartilage (-15%; P < 0.001) than non-TKAs. Longitudinal cartilage thickness change was significantly greater in TKAs than in non-TKAs in the total femorotibial joint (area under the curve [AUC] 0.64), the lateral compartment (AUC 0.66), both tibiae (AUC ≥ 0.61), and the first 9 (of 16) ordered values of subregion change (AUC 0.64-0.69). Discrimination was stronger for TKAs that occurred at 24 and 36 months (n = 18) than for those at 48 and 60 months (n = 22).ConclusionKnees with incident TKA displayed smaller baseline cartilage thickness and greater lateral as well as location-independent ordered value femorotibial cartilage loss than non-TKAs. Discrimination of cartilage loss was greater for TKAs occurring within 2 years after the measurement than for those occurring later.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human cancers and shows resistance to any therapeutic strategy used. Here we tested small-molecule inhibitors targeting chromatin regulators as possible therapeutic agents in PDAC. We show that JQ1, an inhibitor of the bromodomain and extraterminal (BET) family of proteins, suppresses PDAC development in mice by inhibiting both MYC activity and inflammatory signals. The histone deacetylase (HDAC) inhibitor SAHA synergizes with JQ1 to augment cell death and more potently suppress advanced PDAC. Finally, using a CRISPR-Cas9-based method for gene editing directly in the mouse adult pancreas, we show that de-repression of p57 (also known as KIP2 or CDKN1C) upon combined BET and HDAC inhibition is required for the induction of combination therapy-induced cell death in PDAC. SAHA is approved for human use, and molecules similar to JQ1 are being tested in clinical trials. Thus, these studies identify a promising epigenetic-based therapeutic strategy that may be rapidly implemented in fatal human tumors.