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Abstract IS1111 and IS110 insertion sequence (IS) family members encode an unusual DEDD transposase type and exhibit specific target site selection. The IS1111 group include identifiable subterminal inverted repeats (sTIR) not found in the IS110 type1. IS in both families include a noncoding region (NCR) of significant length and, as each individual IS or group of closely related IS selects a different site, we had previously proposed that an NCR-derived RNA was involved in target selection2. Here, we find that the NCR is usually downstream of the transposase gene in IS1111 family IS and upstream in the IS110 type. Four IS1111 and one IS110 family members that target different sequences are used to demonstrate that the NCR determines a short seeker RNA (seekRNA) that co-purified with the transposase. The seekRNA is essential for transposition of the IS or a cargo flanked by IS ends from and to the preferred target. Short sequences matching both top and bottom strands of the target are present in the seekRNA but their order in IS1111 and IS110 family IS is reversed. Reprogramming the seekRNA and donor flank to target a different site is demonstrated, indicating future biotechnological potential for these systems.

Abstract Strategies to fabricate microvascular networks that structurally and functionally mimic native microvessels are needed to address a host of clinical conditions associated with tissue ischemia. The objective of this work was to advance a novel ultrasound technology to fabricate complex, functional microvascular networks directly in vivo. Acoustic patterning utilizes forces within an ultrasound standing wave field (USWF) to organize cells or microparticles volumetrically into defined geometric assemblies. A dual-transducer system was developed to generate USWFs site-specifically in vivo through interference of two ultrasound fields. The system rapidly patterned injected cells or microparticles into parallel sheets within collagen hydrogels in vivo. Acoustic patterning of injected endothelial cells within flanks of immunodeficient mice gave rise to perfused microvessels within 7 days of patterning, whereas non-patterned cells did not survive. Thus, externally-applied ultrasound fields guided injected endothelial cells to self-assemble into perfused microvascular networks in vivo. These studies advance acoustic patterning towards in vivo tissue engineering by providing the first proof-of-concept demonstration that non-invasive, ultrasound-mediated cell patterning can be used to fabricate functional microvascular networks directly in vivo.

Abstract Background Pancreatic ductal adenocarcinoma (PDAC) is a devastating malignancy with a 5-year survival rate of 6% following a diagnosis, and novel therapeutic modalities are needed. Protease-activated receptor 1 (PAR1) is abundantly overexpressed by both tumor cells and multiple stroma cell subsets in the tumor microenvironment (TME), thereby offering a suitable immunotherapy target. Methods A chimeric antigen receptor (CAR) strategy was applied to target PAR1 using a human anti-PAR1 scFv antibody fused to the transmembrane region with two co-stimulatory intracellular signaling domains of cluster of differentiation 28 (CD28) and CD137 (4-1BB), added to CD3ζ in tandem. Results The engineered PAR1CAR-T cells eliminated PAR1 overexpression and transforming growth factor (TGF)-β-mediated PAR1-upregulated cancer cells by approximately 80% in vitro. The adoptive transfer of PAR1CAR-T cells was persistently enhanced and induced the specific regression of established MIA PaCa-2 cancer cells by > 80% in xenograft models. Accordingly, proinflammatory cytokines/chemokines increased in CAR-T-cell-treated mouse sera, whereas Ki67 expression in tumors decreased. Furthermore, the targeted elimination of PAR1-expressing tumors reduced matrix metalloproteinase 1 (MMP1) levels, suggesting that the blocking of the PAR1/MMP1 pathway constitutes a new therapeutic option for PDAC treatment. Conclusions Third-generation PAR1CAR-T cells have antitumor activity in the TME, providing innovative CAR-T-cell immunotherapy against PDAC.

ABSTRACT: Objectives: The aim of this study was to characterise an early clinical multiply antibiotic resistant Acinetobacter baumannii global clone 1 (GC1) isolate from Africa. Methods: The draft genome sequence was determined using short-read (Illumina MiSeq) sequence data and compared to other early GC1 isolates. Resistance genes and other features were identified using various bioinformatics tools. Plasmids were visualised. Results: LUH6050, recovered in South Africa between January 1997 and January 1999, is ST1IP:ST231Ox:KL1:OCL1. Several antibiotic resistance genes (aacC1, aadA2, aphA1, catA1, sul1, and tetA(A)) reside in AbaR32. LUH6050 also includes the plasmid pRAY*, carrying the aadB gentamicin and tobramycin resistance gene, and a 29.9 kb plasmid, pLUH6050-3, carrying the msrE-mphE (macrolide resistance) and dfrA44 (trimethoprim resistance) genes and a small cryptic Rep_1 plasmid. Plasmid pLUH6050-3, a cointegrate of pA1-1 (R3-T1; RepAci1) with an R3-T33 type plasmid encoding a different Rep_3 family Rep, carries 15 pdif sites and 13 dif modules, including those that carry the mrsE-mphE and dfrA44 genes and three that include toxin-antitoxin gene pairs. The closest relative of pLUH6050-3 found in GenBank was from an unrelated 2013 Tanzanian A. baumannii isolate. The chromosome has an AbaR0-type region in comM and includes no ISAba1 copies. Similar features were found in most other sequenced lineage 1 GC1 isolates recovered prior to 2000. Conclusion: LUH6050 represents an early form of the GC1 lineage 1, supplementing limited information about early isolates and isolates from Africa. These data contribute to the understanding of the emergence, evolution, and dissemination of the A. baumannii GC1 clonal complex.

Objectives Vaccinations are an important preventative measure in reducing the spread of infectious diseases worldwide. However, concerns of undervaccination during childhood have become increasingly common. The current study aims to investigate changes in attitudes towards childhood vaccinations prior to the COVID-19 pandemic using a national sample from New Zealand.Design Age-based, period-based, and cohort-based changes were assessed using cohort-sequential latent growth modelling in 11 overlapping birth cohorts, which spanned the ages of 23–79 years.Setting and participants Data were taken from the New Zealand Attitudes and Values Study where 58 654 adults completed at least one wave across a 7-year period (2013 and 2015–2019).Results The period-based and cohort-based models fit the data equally well (χ2(282)=8547.93, p

Development of factor VIII (FVIII) inhibitors is a serious complication in the treatment of hemophilia A (HemA) patients. In clinical trials, anti-CD3 antibody therapy effectively modulates the immune response of allograft rejection or autoimmune diseases without eliciting major adverse effects. In this study, we delivered mRNA-encapsulated lipid nanoparticles (LNPs) encoding therapeutic anti-CD3 antibody (αCD3 LNPs) to overcome the anti-FVIII immune responses in HemA mice. It was found that αCD3 LNPs encoding the single-chain antibodies (Fc-scFv) can efficiently deplete CD3+ and CD4+ effector T cells, whereas αCD3 LNPs encoding double-chain antibodies cannot. Concomitantly, mice treated with αCD3 (Fc-scFv) LNPs showed an increase in the CD4+CD25+Foxp3+ regulatory T cell percentages, which modulated the anti-FVIII immune responses. All T cells returned to normal levels within 2 months. HemA mice treated with αCD3 LNPs prior to hydrodynamic injection of liver-specific FVIII plasmids achieved persistent FVIII gene expression without formation of FVIII inhibitors. Furthermore, transgene expression was increased and persistent following secondary plasmid challenge, indicating induction of long-term tolerance to FVIII. Moreover, the treated mice maintained their immune competence against other antigens. In conclusion, our study established a potential new strategy to induce long-term antigen-specific tolerance using an αCD3 LNP formulation.

Abstract Background Bioactive lipids play an important role in insulin secretion and sensitivity, contributing to the pathophysiology of type 2 diabetes (T2D). This study aimed to identify novel lipid species associated with incident T2D in a nested case–control study within a long-term prospective Chinese community-based cohort with a median follow-up of ~ 16 years. Methods Plasma samples from 196 incident T2D cases and 196 age- and sex-matched non-T2D controls recruited from the Hong Kong Cardiovascular Risk Factor Prevalence Study (CRISPS) were first analyzed using untargeted lipidomics. Potential predictive lipid species selected by the Boruta analysis were then verified by targeted lipidomics. The associations between these lipid species and incident T2D were assessed. Effects of novel lipid species on insulin secretion in mouse islets were investigated. Results Boruta analysis identified 16 potential lipid species. After adjustment for body mass index (BMI), triacylglycerol/high-density lipoprotein (TG/HDL) ratio and the presence of prediabetes, triacylglycerol (TG) 12:0_18:2_22:6, TG 16:0_11:1_18:2, TG 49:0, TG 51:1 and diacylglycerol (DG) 18:2_22:6 were independently associated with increased T2D risk, whereas lyso-phosphatidylcholine (LPC) O-16:0, LPC P-16:0, LPC O-18:0 and LPC 18:1 were independently associated with decreased T2D risk. Addition of the identified lipid species to the clinical prediction model, comprised of BMI, TG/HDL ratio and the presence of prediabetes, achieved a 3.8% improvement in the area under the receiver operating characteristics curve (AUROC) (p = 0.0026). Further functional study revealed that, LPC O-16:0 and LPC O-18:0 significantly potentiated glucose induced insulin secretion (GSIS) in a dose-dependent manner, whereas neither DG 18:2_22:6 nor TG 12:0_18:2_22:6 had any effect on GSIS. Conclusions Addition of the lipid species substantially improved the prediction of T2D beyond the model based on clinical risk factors. Decreased levels of LPC O-16:0 and LPC O-18:0 may contribute to the development of T2D via reduced insulin secretion.

Abstract Background Olfactory dysfunction (OD) is associated with both post‐viral and inflammatory etiologies such as COVID‐19 and chronic rhinosinusitis/rhinitis (CRS/R) respectively, to result in reduced quality of life (QoL). However, the former typically induces a sudden‐onset OD while the latter has a gradual presentation. This study aims to establish and compare health utility values (HUVs) and olfactory‐specific QoL measurements between patients with COVID‐19 and CRS/R related OD. Methods This prospective study surveyed COVID‐19 and CRS/R patients with self‐reported OD using HUV assessments (EuroQol‐visual analog scale [EQ‐VAS], EuroQol‐5 dimension [EQ‐5D], time trade‐off [TTO]) and olfactory and sinonasal QoL measures (questionnaire of olfactory disorders –negative and positive statements [QOD‐NS + PS] and sino‐nasal outcome test [SNOT‐22]). A subgroup of subjects completed objective olfactory testing. Intergroup mean scores were compared using Mann–Whitney U tests. Results One hundred eleven subjects were enrolled: mean age ± SD (43.0 ± 15.4 years), 55.9% female. CRS/R was associated with lower HUVs as measured by EQ‐VAS (CRS/R: 0.67 ± 0.18 vs. COVID‐19: 0.74 ± 0.19, p = .03) and worse SNOT‐22 scores in both overall (CRS/R: 49.03 ± 21.04 vs. COVID‐19: 27.58 ± 18.45, p

ABSTRACT The insertion sequence IS26 plays a key role in the spread of antibiotic resistance genes in Gram-negative bacteria. IS26 and members of the IS26 family are able to use two distinct mechanisms to form cointegrates made up of two DNA molecules linked via directly oriented copies of the IS. The well-known copy-in (formerly replicative) reaction occurs at very low frequency, and the more recently discovered targeted conservative reaction, which joins two molecules that already include an IS, is substantially more efficient. Experimental evidence has indicated that, in the targeted conservative mode, the action of Tnp26, the IS26 transposase, is required only at one end. How the Holliday junction (HJ) intermediate generated by the Tnp26-catalyzed single-strand transfer is processed to form the cointegrate is not known. We recently proposed that branch migration and resolution via the RuvABC system may be needed to process the HJ; here, we have tested this hypothesis. In reactions between a wild-type and a mutant IS26, the presence of mismatched bases near one IS end impeded the use of that end. In addition, evidence of gene conversion, potentially consistent with branch migration, was detected in some of the cointegrates formed. However, the targeted conservative reaction occurred in strains that lacked the recG, ruvA, or ruvC genes. As the RuvC HJ resolvase is not required for targeted conservative cointegrate formation, the HJ intermediate formed by the action of Tnp26 must be resolved by an alternate route. IMPORTANCE In Gram-negative bacteria, the contribution of IS26 to the spread of antibiotic resistance and other genes that provide cells with an advantage under specific conditions far exceeds that of any other known insertion sequence. This is likely due to the unique mechanistic features of IS26 action, particularly its propensity to cause deletions of adjacent DNA segments and the ability of IS26 to use two distinct reaction modes for cointegrate formation. The high frequency of the unique targeted conservative reaction mode that occurs when both participating molecules include an IS26 is also key. Insights into the detailed mechanism of this reaction will help to shed light on how IS26 contributes to the diversification of the bacterial and plasmid genomes it is found in. These insights will apply more broadly to other members of the IS26 family found in Gram-positive as well as Gram-negative pathogens.

Gene therapy offers great promises for a cure of hemophilia A resulting from factor VIII (FVIII) gene deficiency. We have developed and optimized a non-viral ultrasound-mediated gene delivery (UMGD) strategy. UMGD of reporter plasmids targeting mice livers achieved high levels of transgene expression predominantly in hepatocytes. Following UMGD of a plasmid encoding human FVIII driven by a hepatocyte-specific promoter/enhancer (pHP-hF8/N6) into the livers of hemophilia A mice, a partial phenotypic correction was achieved in treated mice. In order to achieve persistent and therapeutic FVIII gene expression, we adopted a plasmid (pHP-hF8-X10) encoding an FVIII variant with significantly increased FVIII secretion. By employing an optimized pulse-train ultrasound condition and immunomodulation, the treated hemophilia A mice achieved 25%–150% of FVIII gene expression on days 1–7 with very mild transient liver damage, as indicated by a small increase of transaminase levels that returned to normal within 3 days. Therapeutic levels of FVIII can be maintained persistently without the generation of inhibitors in mice. These results indicate that UMGD can significantly enhance the efficiency of plasmid DNA transfer into the liver. They also demonstrate the potential of this novel technology to safely and effectively treat hemophilia A.

Abstract Background Resistance to anti-malarials is a serious threat to the efforts to control and eliminate malaria. Surveillance based on simple field protocols with centralized testing to detect molecular markers associated with anti-malarial drug resistance can be used to identify locations where further investigations are needed. Methods Dried blood spots were collected from 398 patients (age range 5–59 years, 99% male) with Plasmodium falciparum infections detected using rapid diagnostic tests over two rounds of sample collection conducted in 2016 and 2017 in Komé, South-West Chad. Specimens were genotyped using amplicon sequencing or qPCR for validated markers of anti-malarial resistance including partner drugs used in artemisinin-based combination therapy (ACT). Results No mutations in the pfk13 gene known to be associated with artemisinin resistance were found but a high proportion of parasites carried other mutations, specifically K189T (190/349, 54.4%, 95%CI 49.0–59.8%). Of 331 specimens successfully genotyped for pfmdr1 and pfcrt, 52% (95%CI 46.4–57.5%) carried the NFD-K haplotype, known to be associated with reduced susceptibility to lumefantrine. Only 20 of 336 (6.0%, 95%CI 3.7–9.0%) had parasites with the pfmdr1-N86Y polymorphism associated with increased treatment failures with amodiaquine. Nearly all parasites carried at least one mutation in pfdhfr and/or pfdhps genes but ‘sextuple’ mutations in pfdhfr—pfdhps including pfdhps -A581G were rare (8/336 overall, 2.4%, 95%CI 1.2–4.6%). Only one specimen containing parasites with pfmdr1 gene amplification was detected. Conclusions These results provide information on the likely high efficacy of artemisinin-based combinations commonly used in Chad, but suggest decreasing levels of sensitivity to lumefantrine and high levels of resistance to sulfadoxine-pyrimethamine used for seasonal malaria chemoprevention and intermittent preventive therapy in pregnancy. A majority of parasites had mutations in the pfk13 gene, none of which are known to be associated with artemisinin resistance. A therapeutic efficacy study needs to be conducted to confirm the efficacy of artemether-lumefantrine.

Aspergillus fungi produce mycotoxins that are detrimental to human and animal health. Two sections of aspergilli are of particular importance to cereal food crops such as corn and barley. Aspergillus section Flavi species like A. flavus and A. parasiticus produce aflatoxins, while section Circumdati species like A. ochraceus and A. sclerotiorum produce ochratoxin A. Mitigating these toxins in food and feed is a critical and ongoing worldwide effort. We have previously investigated biosynthetic gene clusters in Aspergillus flavus that are linked to fungal virulence in corn. We found that one such cluster, asa, is responsible for the production of aspergillic acid, an iron-binding, hydroxamic acid-containing pyrazinone metabolite. Furthermore, we found that the asa gene cluster is present in many other aflatoxin- and ochratoxin-producing aspergilli. The core gene in the asa cluster encodes the small nonribosomal peptide synthetase-like (NRPS-like) protein AsaC. We have swapped the asaC ortholog from A. sclerotiorum into A. flavus, replacing its native copy, and have also cloned both asaC orthologs into Saccharomyces cerevisiae. We show that AsaC orthologs in section Flavi and section Circumdati, while only containing adenylation-thiolation-reductase (ATR) domains, can selectively biosynthesize distinct pyrazinone natural products: deoxyaspergillic acid and flavacol, respectively. Because pyrazinone natural products and the gene clusters responsible for their production are implicated in a variety of important microbe-host interactions, uncovering the function and selectivity of the enzymes involved could lead to strategies that ultimately benefit human health.

Abstract Objectives Malaria infection is still known to be a worldwide public health problem, especially in tropical and sub-tropical African countries like Sudan. A pilot study conducted to describe the trend of P. falciparum drug resistance markers in 2017–2018 in comparison to CQ and AS/SP eras in Sudan. The Pfcrt, Pfmdr-1, Pfdhfr, and Pfdhps genes were investigated. Data deposited by the worldwide antimalarial resistance network was consulted, and the molecular markers previously reported from Sudan were analyzed. Results Drug molecular markers analysis was successfully done on 20 P. falciparum isolates. The Pfcrt K76 showed high frequency; 16 (80%). For the Pfmdr-1, 9 (45%) isolates were carrying the N86 allele, and 11 (55%) were 86Y allele. While the Y184F of the Pfmdr-1 showed a higher frequency of 184F compared to Y184; 16 (80%) and 4 (20%), respectively. In the Pfdhfr, 51I allele showed higher frequency compared to N51; 18 (90%) and 2 (10%), respectively. For S108N, 18 (90%) were 108 N and 2 (10%) were S108. In the Pfdhps, all isolates were carrying the mutant alleles; 437G and 540E. The frequency distribution of the Pfcrt, Pfmdr-1, Pfdhfr, Pfdhps was significantly different across the whole years in Sudan.

Understanding how SARS-CoV-2 gains initial entry into the human body is a key step towards the development of prophylaxes and therapeutics for COVID-19. Here, the authors show that ACE2, the receptor for SARS-CoV-2, is abundantly expressed in the motile cilia of the human nasal and respiratory tract and is not affected by the use of angiotensin-converting enzyme inhibitors or angiotensin II receptor blockers.

Hemophilia A (HemA) patients are currently treated with costly and inconvenient replacement therapy of short-lived factor VIII (FVIII) protein. Development of lipid nanoparticle (LNP)-encapsulated mRNA encoding FVIII can change this paradigm. LNP technology constitutes a biocompatible and scalable system to efficiently package and deliver mRNA to the target site. Mice intravenously infused with the luciferase mRNA LNPs showed luminescence signals predominantly in the liver 4 h after injection. Repeated injections of LNPs did not induce elevation of liver transaminases. We next injected LNPs carrying mRNAs encoding different variants of human FVIII (F8 LNPs) into HemA mice. A single injection of B domain-deleted F8 LNPs using different dosing regimens achieved a wide range of therapeutic activities rapidly, which can be beneficial for various usages in hemophilia treatment. The expression slowly declined yet remained above therapeutic levels up to 5–7 days post-injection. Furthermore, routine repeated injections of F8 LNPs in immunodeficient mice produced consistent expression of FVIII over time. In conclusion, F8 LNP treatment produced rapid and prolonged duration of FVIII expression that could be applied to prophylactic treatment and potentially various other treatment options. Our study showed potential for a safe and effective platform of new mRNA therapies for HemA.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a 5-year survival rate of 9%. Major obstacles to successful treatment of pancreatic cancer are the immunosuppressive tumor microenvironment (TME) and antigenic complexity or heterogeneity. Programmed death-ligand 1 (PD-L1) is expressed on PDAC and immunosuppressed cells within the TME, providing suitable immunotherapy targets. We applied a chimeric antigen receptor (CAR) strategy to target immune checkpoint programmed death-1 (PD-1)/PD-L1 interactions. Lentiviral vectors were used to express the extracellular domain of human PD-1 (PD-1-CD28-4-1BB activating chimeric receptor [PD1ACR]) or the single-chain variable fragment (scFv) region of anti-PD-L1 (PDL1CAR) that binds to PD-L1, and each was fused to intracellular signaling domains containing CD3 zeta, CD28, and 4-1BB (CD137). Both engineered CAR T cells recognized and eliminated PD-L1-overexpressing CFPAC1 cells efficiently at approximately 80% in vitro. Adoptive transfer of both CAR T cells enhanced T cell persistence and induced specific regression of established CFPAC1 cancer by >80% in both xenograft and orthotopic models. Ki67 expression in tumors decreased, whereas proinflammatory cytokines/chemokines increased in CAR T cell-treated mouse sera. PD1ACR and PDL1CAR obtained a similar therapeutic efficacy. Thus, these armed third-generation PD-L1-targeted CAR T cells confer antitumor activity and the ability to combat T cell exhaustion, providing a potentially new and innovative CAR T cell immunotherapy against pancreatic cancers.

Abstract Background Olfactory dysfunction is a prevalent problem with a significant impact on quality of life and increased mortality. Limited effective therapies exist. Platelet‐rich plasma (PRP) is an autologous biologic product with anti‐inflammatory and neuroprotective effects. This novel pilot study evaluated the role of PRP on olfactory neuroregeneration in patients with hyposmia. Methods Seven patients who had olfactory loss greater than 6 months in duration, no evidence of sinonasal inflammatory disease, and no improvement with olfactory training and budesonide topical rinses were enrolled in this preliminary study. Patients received a single intranasal injection of PRP into the mucosa of the olfactory cleft. The Sniffin' Sticks olfactory test consisting of threshold, discrimination, and identification measurements (TDI) was administered at the beginning of the study and at 1 and 3 months. Results All patients reported a subjective improvement of their smell shortly after injection but then stabilized. At 3‐month post‐treatment, two patients with functional anosmia (TDI 16 but 30) at 3‐month follow‐up. On average, patients with baseline TDI > 16 improved by 5.85 points with the most significant improvement in the threshold subcomponent. There were no adverse outcomes from intranasal PRP injections. Conclusion PRP appears safe for use in the treatment of olfactory loss, and preliminary data suggest possible efficacy, especially for those with moderate yet persistent loss. Further studies will help determine optimal frequency and duration of use. Level of evidence: 2B

Aspergillus flavus is an opportunistic fungal pathogen capable of producing aflatoxins, potent carcinogenic toxins that accumulate in maize kernels after infection. To better understand the molecular mechanisms of maize resistance to A. flavus growth and aflatoxin accumulation, we performed a high-throughput transcriptomic study in situ using maize kernels infected with A. flavus strain 3357. Three maize lines were evaluated: aflatoxin-contamination resistant line TZAR102, semi-resistant MI82, and susceptible line Va35. A modified genotype-environment association method (GEA) used to detect loci under selection via redundancy analysis (RDA) was used with the transcriptomic data to detect genes significantly influenced by maize line, fungal treatment, and duration of infection. Gene ontology enrichment analysis of genes highly expressed in infected kernels identified molecular pathways associated with defense responses to fungi and other microbes such as production of pathogenesis-related (PR) proteins and lipid bilayer formation. To further identify novel genes of interest, we incorporated genomic and phenotypic field data from a genome wide association analysis with gene expression data, allowing us to detect significantly expressed quantitative trait loci (eQTL). These results identified significant association between flavonoid biosynthetic pathway genes and infection by A. flavus. In planta fungal infections showed that the resistant line, TZAR102, has a higher fold increase of the metabolites naringenin and luteolin than the susceptible line, Va35, when comparing untreated and fungal infected plants. These results suggest flavonoids contribute to plant resistance mechanisms against aflatoxin contamination through modulation of toxin accumulation in maize kernels.

Summary: Understanding viral tropism is an essential step toward reducing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission, decreasing mortality from coronavirus disease 2019 (COVID-19) and limiting opportunities for mutant strains to arise. Currently, little is known about the extent to which distinct tissue sites in the human head and neck region and proximal respiratory tract selectively permit SARS-CoV-2 infection and replication. In this translational study, we discover key variabilities in expression of angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2), essential SARS-CoV-2 entry factors, among the mucosal tissues of the human proximal airways. We show that SARS-CoV-2 infection is present in all examined head and neck tissues, with a notable tropism for the nasal cavity and tracheal mucosa. Finally, we uncover an association between smoking and higher SARS-CoV-2 viral infection in the human proximal airway, which may explain the increased susceptibility of smokers to developing severe COVID-19. This is at least partially explained by differences in interferon (IFN)-β1 levels between smokers and non-smokers.

Human plasma fibronectin is an adhesive protein that plays a crucial role in wound healing. Many studies had indicated that glycans might mediate the expression and functions of fibronectin, yet a comprehensive understanding of its glycosylation is still missing. Here, we performed a comprehensive N- and O-glycosylation mapping of human plasma fibronectin and quantified the occurrence of each glycoform in a site-specific manner. Intact N-glycopeptides were enriched by zwitterionic hydrophilic interaction chromatography, and N-glycosite sites were localized by the 18O-labeling method. O-glycopeptide enrichment and O-glycosite identification were achieved by an enzyme-assisted site-specific extraction method. An RP–LC–MS/MS system functionalized with collision-induced dissociation and stepped normalized collision energy (sNCE)-HCD tandem mass was applied to analyze the glycoforms of fibronectin. A total of 6 N-glycosites and 53 O-glycosites were identified, which were occupied by 38 N-glycoforms and 16 O-glycoforms, respectively. Furthermore, 77.31% of N-glycans were sialylated, and O-glycosylation was dominated by the sialyl-T antigen. These site-specific glycosylation patterns on human fibronectin can facilitate functional analyses of fibronectin and therapeutics development.

Abstract Parasite resistance against anti-malarial drugs is a major threat to the ongoing malaria control and elimination strategies. This is especially true since resistance to the currently recommended artemisinins and partner drugs has been confirmed in South East Asia (SEA) and new anti-malarial compounds are not expected to be available in the near future. Spread from SEA or independent emergence of artemisinin resistance in sub-Saharan Africa (SSA) could reverse the achievements in malaria control that have been attained in the past two decades and derail the ongoing elimination strategies. The current surveillance of clinical efficacy and resistance to anti-malarial drugs is based on efficacy trials to assess the clinical performance of anti-malarials, in vivo/ex vivo assessment of parasite susceptibility to anti-malarials and prevalence of known molecular markers of drug resistance. Whereas clinical efficacy trials are restricted by cost and the complex logistics of patient follow-up, molecular detection of genetic mutations associated with resistance or reduced susceptibility to anti-malarials is by contrast a simple and powerful tool for early detection and monitoring of the prevalence of resistant parasites at population level. This provides needed information before clinical failure emerges, allowing policy makers to anticipate problems and respond. The various methods previously used in detection of molecular markers of drug resistance share some limitations: low-throughput, and high costs per sample and demanding infrastructure. However, recent technological advances including next-generation sequencing (NGS) methodologies promise greatly increased throughput and reduced costs, essentially providing unprecedented potential to address different research and operational questions of relevance for drug policy. This review assesses the potential role of NGS to provide comprehensive information that could guide drug policies in malaria endemic countries and looks at the foreseeable challenges facing the establishment of NGS approaches for routine surveillance of parasite resistance to anti-malarials in SSA.

Abstract Background Malaria causes a reduction in haemoglobin that is compounded by primaquine, particularly in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. The aim of this study was to determine the relative contributions to red cell loss of malaria and primaquine in patients with uncomplicated Plasmodium vivax. Methods A systematic review identified P. vivax efficacy studies of chloroquine with or without primaquine published between January 2000 and March 2017. Individual patient data were pooled using standardised methodology, and the haematological response versus time was quantified using a multivariable linear mixed effects model with non-linear terms for time. Mean differences in haemoglobin between treatment groups at day of nadir and day 42 were estimated from this model. Results In total, 3421 patients from 29 studies were included: 1692 (49.5%) with normal G6PD status, 1701 (49.7%) with unknown status and 28 (0.8%) deficient or borderline individuals. Of 1975 patients treated with chloroquine alone, the mean haemoglobin fell from 12.22 g/dL [95% CI 11.93, 12.50] on day 0 to a nadir of 11.64 g/dL [11.36, 11.93] on day 2, before rising to 12.88 g/dL [12.60, 13.17] on day 42. In comparison to chloroquine alone, the mean haemoglobin in 1446 patients treated with chloroquine plus primaquine was − 0.13 g/dL [− 0.27, 0.01] lower at day of nadir (p = 0.072), but 0.49 g/dL [0.28, 0.69] higher by day 42 (p 25% to 5 g/dL. Conclusions Primaquine has the potential to reduce malaria-related anaemia at day 42 and beyond by preventing recurrent parasitaemia. Its widespread implementation will require accurate diagnosis of G6PD deficiency to reduce the risk of drug-induced haemolysis in vulnerable individuals. Trial registration This trial was registered with PROSPERO: CRD42016053312. The date of the first registration was 23 December 2016.

The inflammatory response to viral infection in humans is a dynamic process with complex cell interactions that are governed by the immune system and influenced by both host and viral factors. Due to this complexity, the relative contributions of the virus and host factors are best studied in vivo using animal models. In this review, we describe how the zebrafish (Danio rerio) has been used as a powerful model to study host-virus interactions and inflammation by combining robust forward and reverse genetic tools with in vivo imaging of transparent embryos and larvae. The innate immune system has an essential role in the initial inflammatory response to viral infection. Focused studies of the innate immune response to viral infection are possible using the zebrafish model as there is a 4-6 week timeframe during development where they have a functional innate immune system dominated by neutrophils and macrophages. During this timeframe, zebrafish lack a functional adaptive immune system, so it is possible to study the innate immune response in isolation. Sequencing of the zebrafish genome has revealed significant genetic conservation with the human genome, and multiple studies have revealed both functional conservation of genes, including those critical to host cell infection and host cell inflammatory response. In addition to studying several fish viruses, zebrafish infection models have been developed for several human viruses, including influenza A, noroviruses, chikungunya, Zika, dengue, herpes simplex virus type 1, Sindbis, and hepatitis C virus. The development of these diverse viral infection models, coupled with the inherent strengths of the zebrafish model, particularly as it relates to our understanding of macrophage and neutrophil biology, offers opportunities for far more intensive studies aimed at understanding conserved host responses to viral infection. In this context, we review aspects relating to the evolution of innate immunity, including the evolution of viral pattern recognition receptors, interferons and interferon receptors, and non-coding RNAs.

Previously, authors reported that individual volatile organic compounds (VOCs) emitted by non-aflatoxigenic Aspergillus flavus could act as a mechanism of biocontrol to significantly reduce aflatoxins and cyclopiazonic acid (CPA) produced by toxigenic strains. In this study, various combinations and volumes of three mycotoxin-reductive VOCs (2,3-dihydrofuran, 3-octanone and decane) were assessed for their cumulative impacts on four Aspergillus strains (LA1–LA4), which were then analyzed for changes in growth, as well as the production of mycotoxins, including aflatoxins, CPA and multiple indole diterpenes. Fungal growth remained minimally inhibited when exposed to various combinations of VOCs. No single combination was able to consistently, or completely, inhibit aflatoxin or CPA across all toxigenic strains tested. However, the combination of 2,3-dihydrofuran and 3-octanone offered the greatest overall reductions in aflatoxin and CPA production. Despite no elimination of their production, findings showed that combining VOCs produced solely by non-aflatoxigenic A. flavus still inhibited several agriculturally important mycotoxins, including B and G aflatoxins and CPA. Therefore, other VOC combinations are worth testing as post-harvest biocontrol treatments to ensure the prolonged effectiveness of pre-harvest biocontrol efforts.

Introduction Investigate the effects of switching from two times per day exenatide to once-weekly exenatide administered by autoinjector (exenatide once-weekly suspension by autoinjector (QWS-AI)) or treatment with exenatide QWS-AI for 1 year.Research design and methods In this phase III open-label study, adults with type 2 diabetes were randomized to receive exenatide QWS-AI (2 mg) or exenatide two times per day (5 mcg for 4 weeks, followed by 10 mcg) for 28 weeks. During a subsequent non-randomized 24-week extension, patients who received exenatide two times per day were switched to exenatide QWS-AI and those randomized to exenatide QWS-AI continued this treatment. Efficacy measures included changes from baseline in glycated hemoglobin (A1C), fasting plasma glucose (FPG), and body weight.Results In total, 315 patients (mean baseline A1C of 8.5%) completed the initial 28 weeks of randomized treatment with exenatide QWS-AI (n=197) or exenatide two times per day (n=118) and were included in the 24-week extension (mean A1C of 7.0% and 7.3%, respectively, at week 28). From weeks 28–52, patients who switched from exenatide two times per day to exenatide QWS-AI had additional A1C reductions of approximately 0.5% (mean A1C change from baseline of –1.4% at week 52) and further reductions from baseline in FPG. Patients who continued exenatide QWS-AI treatment for 52 weeks showed clinically relevant A1C reductions (mean A1C change from baseline of –1.3% at week 52). Body-weight reductions achieved through week 28 were sustained at week 52 in both groups. There were no unexpected safety concerns or changes in the safety profile among patients who switched from exenatide two times per day to exenatide QWS-AI or those who continued exenatide QWS-AI treatment for 52 weeks.Conclusions Switching from exenatide two times per day to exenatide QWS-AI resulted in further A1C reductions and maintenance of earlier decreases in body weight, while continued therapy with exenatide QWS-AI for 52 weeks maintained A1C and body-weight reductions, without additional safety or tolerability concerns.Trial registration number NCT01652716.

Hemophilia A is a genetic disorder that results in the deficiency of functional factor VIII protein, which plays a key role in blood coagulation. Currently, the majority of hemophilia A patients are treated with repeated infusions of factor VIII protein. Approximately 30% of severe hemophilia A patients develop neutralizing antibodies to factor VIII (known as factor VIII inhibitors) due to treatment, rendering factor VIII protein infusions ineffective. Previously, mice receiving murine IL-2 complexed with α-murine IL-2 mAbs (JES6-1A12) showed a lack of factor VIII inhibitor formation after factor VIII treatment, which was associated with the proliferation and the activation of factor VIII-specific regulatory T cells (Tregs). In this paper, we evaluated if an Fc-fused mutated protein analog of mouse IL-2, named Fc.Mut24, engineered to selectively promote the expansion of Tregs in vivo can modulate factor VIII-specific immune responses. The mice received one intraperitoneal injection of Fc.Mut24. When the regulatory T cell population reached its highest frequency and peak activation, the mice received a hydrodynamic injection of factor VIII plasmid (day 4) followed by a second Fc.Mut24 dose (day 7). Peripheral blood was collected weekly. Flow cytometry was used to characterize the peripheral blood cell populations, while ELISA and Bethesda assays were used to assess the inhibitor concentrations and the functional titers in plasma. The activated partial thromboplastin time assay was used to assess the functional activities of factor VIII in blood. The mice receiving Fc.Mut24 showed a dramatic and transient increase in the population of activated Tregs after Fc.Mut24 injection. Factor VIII gene therapy via hydrodynamic injection resulted in high anti-factor VIII inhibitor concentrations in control PBS-injected mice, whereas the mice treated with Fc.Mut24 produced no inhibitors. Most significantly, there were no inhibitors generated after a second hydrodynamic injection of factor VIII plasmid administered at 19 weeks after the first injection in Fc.Mut24-treated mice. The mice receiving Fc.Mut24 maintained high levels of factor VIII activity throughout the experiment, while the control mice had the factor VIII activity dropped to undetectable levels a few weeks after the first factor VIII plasmid injection. Our data show that human therapies analogous to Fc.Mut24 could potentially provide a method to prevent inhibitor formation and induce long-term immune tolerance to factor VIII in hemophilia patients.

Human coagulation factor VIII (FVIII) is a key co-factor in the clotting cascade, the deficiency of which leads to Hemophilia A. Human plasma-derived (pdFVIII) and recombinant FVIII (rFVIII) had been used as effective products to prevent and treat bleeding episodes. Both FVIII products share identical amino acid sequences and appear to be equivalent as of clinical efficiency. However, systemic reviews found an increased risk of neutralizing antibody (or inhibitor) development with recombinant products. FVIII is a highly glycosylated protein, and its glycosylation pattern is specific to host cells and environments. The roles of glycosylation in immune responses toward pdFVIII and rFVIII are yet to be defined. Herein, we systemically profiled N- and O-glycomes of pdFVIII and rFVIII using a mass spectrometry-based glycoproteomic strategy. A total of 110 site-specific N-glycopeptides consisting of 61 N-glycoforms were identified quantitatively from rFVIII and pdFVIII. Additionally, 31 O-glycoforms were identified on 23 peptides from rFVIII and pdFVIII. A comprehensive comparison of their site-specific glycan profiles revealed distinct differences between the glycosylation of pdFVIII and rFVIII.

Case summary A 9-year-old male neutered Maine Coon cat presented with a 6-month history of polyphagia and one recent episode of tremors and weakness. Blood work revealed profound hypoglycemia and results of a paired insulin glucose test were consistent with an insulinoma. Abdominal ultrasound revealed a solitary pancreatic mass, and results of a fine-needle aspirate (FNA) gave further support for the location of the neuroendocrine tumor. After unsuccessful medical management of the hypoglycemia, the mass was surgically removed. Immunohistochemistry confirmed that it was an insulinoma. At the time of writing, the patient had been in clinical remission for 9 months. Relevance and novel information Feline insulinomas are rare and there is very little information on their behavior, clinical course and histologic characteristics. This is the first reported case of an insulinoma in a Maine Coon cat and the first to describe results of an ultrasound-guided FNA of the mass. In addition, the progression of disease, histopathology and immunohistochemistry results add to the currently minimal database for feline insulinomas.

We have achieved significant enhancement of gene delivery into livers of large animals using ultrasound (US)-targeted microbubble (MB) destruction methods. An infusion of pGL4 (encoding a luciferase reporter gene) plasmid DNA (pDNA) and MBs into a portal-vein segmental branch of a porcine liver was exposed to US for 4 min. Therapeutic US induced cavitation of MBs to temporarily permeabilize the vascular endothelium and cell membranes, allowing entry of pDNA. We obtained a 64-fold enhancement in luciferase expression in pig livers compared to control without US using an unfocused, dual-element transducer (H105, center frequency [fc] = 1.10 MHz) at 2.7 MPa peak negative pressure (PNP). However, input electrical energy was limited, and modified transducers were designed to have spherical (H185A, fc = 1.10 MHz) or cylindrical foci (H185B, fc = 1.10 MHz; H185D, fc = 1.05 MHz) to enhance PNP output. The revised transducers required less electrical input to achieve 2.7 MPa PNP compared to H105, thereby allowing PNP outputs of up to 6.2 MPa without surpassing the piezo-material limitations. Subsequently, luciferase expression significantly improved up to 9,000-fold compared to controls with minor liver damage. These advancements will allow us to modify our current protocols toward minimally invasive US gene therapy. Keywords: ultrasound, gene delivery, animal studies, liver, luciferase, ultrasound-mediated gene transfer, nonviral gene transfer, focused ultrasound

Abstract Background There exist several predictive risk models for cardiovascular disease (CVD), including some developed specifically for patients with type 2 diabetes mellitus (T2DM). However, the models developed for a diabetic population are based on information derived from medical records or laboratory results, which are not typically available to entities like payers or quality of care organizations. The objective of this study is to develop and validate models predicting the risk of cardiovascular events in patients with T2DM based on medical insurance claims data. Methods Patients with T2DM aged 50 years or older were identified from the Optum™ Integrated Real World Evidence Electronic Health Records and Claims de-identified database (10/01/2006–09/30/2016). Risk factors were assessed over a 12-month baseline period and cardiovascular events were monitored from the end of the baseline period until end of data availability, continuous enrollment, or death. Risk models were developed using logistic regressions separately for patients with and without prior CVD, and for each outcome: (1) major adverse cardiovascular events (MACE; i.e., non-fatal myocardial infarction, non-fatal stroke, CVD-related death); (2) any MACE, hospitalization for unstable angina, or hospitalization for congestive heart failure; (3) CVD-related death. Models were developed and validated on 70% and 30% of the sample, respectively. Model performance was assessed using C-statistics. Results A total of 181,619 patients were identified, including 136,544 (75.2%) without prior CVD and 45,075 (24.8%) with a history of CVD. Age, diabetes-related hospitalizations, prior CVD diagnoses and chronic pulmonary disease were the most important predictors across all models. C-statistics ranged from 0.70 to 0.81, indicating that the models performed well. The additional inclusion of risk factors derived from pharmacy claims (e.g., use of antihypertensive, and use of antihyperglycemic) or from medical records and laboratory measures (e.g., hemoglobin A1c, urine albumin to creatinine ratio) only marginally improved the performance of the models. Conclusion The claims-based models developed could reliably predict the risk of cardiovascular events in T2DM patients, without requiring pharmacy claims or laboratory measures. These models could be relevant for providers and payers and help implement approaches to prevent cardiovascular events in high-risk diabetic patients.

Abstract Background In Niger, malaria transmission is markedly seasonal with most of the disease burden occurring in children during the rainy season. Seasonal malaria chemoprevention (SMC) with amodiaquine plus sulfadoxine–pyrimethamine (AQ + SP) is recommended in the country to be administered monthly just before and during the rainy season. Moreover, clinical decisions on use of SP for intermittent preventive treatment in pregnancy (IPTp) now depend upon the validated molecular markers for SP resistance in Plasmodium falciparum observed in the local parasite population. However, little is known about molecular markers of resistance for either SP or AQ in the south of Niger. To address this question, clinical samples which met clinical and biological criteria, were collected in Gabi, Madarounfa district, Maradi region, Niger in 2011–2012 (before SMC implementation). Molecular markers of resistance to pyrimethamine (pfdhfr), sulfadoxine (pfdhps) and amodiaquine (pfmdr1) were assessed by DNA sequencing. Results Prior to SMC implementation, the samples showed a high proportion of clinical samples that carried the pfdhfr 51I/59R/108N haplotype associated with resistance to pyrimethamine and pfdhps 436A/F/H and 437G mutations associated with reduced susceptibility to sulfadoxine. In contrast mutations in codons 581G, and 613S in the pfdhps gene, and in pfmdr1, 86Y, 184Y, 1042D and 1246Y associated with resistance to amodiaquine, were less frequently observed. Importantly, pfdhfr I164L and pfdhps K540E mutations shown to be the most clinically relevant markers for high level clinical resistance to SP were not detected in Gabi. Conclusions Although parasites with genotypes associated with the highest levels of resistance to AQ + SP are not yet common in this setting, their importance for deployment of SMC and IPTp dictates that monitoring of these markers of resistance should accompany these interventions. This study also highlights the parasite heterogeneity within a small spatial area and the need to use caution when extrapolating results from surveys of molecular markers of resistance in a single site to inform regional policy decisions.

Abstract Background Achieving control of glycated hemoglobin (HbA1c), blood pressure (BP), and body weight (BW) remains a challenge for most patients with type 2 diabetes mellitus (T2DM). In clinical trials, canagliflozin (CANA), an inhibitor of sodium-glucose co-transporter 2, has shown significant improvement compared to some dipeptidyl peptidase-4 (DPP-4) inhibitors in the achievement of such quality measures. This study used recent electronic medical records (EMR) data to assess quality measure achievement of HbA1C, BP, and BW loss in patients treated with CANA versus DPP-4 inhibitors. Methods Adult patients with ≥1 T2DM diagnosis and ≥12 months of clinical activity (baseline) before first CANA or DPP-4 prescription (index) were identified in the QuintilesIMS Health Real-World Data EMRs–US database (03/29/2012–10/30/2015). Patients were observed from the index to last encounter. Inverse probability of treatment weighting (IPTW) was used to adjust for observed baseline confounders between groups. Kaplan-Meier (KM) rates and Cox proportional hazard models were used to compare achievement of HbA1c

Abstract The delivery of safe and effective radical cure for Plasmodium vivax is one of the greatest challenges for achieving malaria elimination from the Asia–Pacific by 2030. During the annual meeting of the Asia Pacific Malaria Elimination Network Vivax Working Group in October 2016, a round table discussion was held to discuss the programmatic issues hindering the widespread use of primaquine (PQ) radical cure. Participants included 73 representatives from 16 partner countries and 33 institutional partners and other research institutes. In this meeting report, the key discussion points are presented and grouped into five themes: (i) current barriers for glucose-6-phosphate deficiency (G6PD) testing prior to PQ radical cure, (ii) necessary properties of G6PD tests for wide scale deployment, (iii) the promotion of G6PD testing, (iv) improving adherence to PQ regimens and (v) the challenges for future tafenoquine (TQ) roll out. Robust point of care (PoC) G6PD tests are needed, which are suitable and cost-effective for clinical settings with limited infrastructure. An affordable and competitive test price is needed, accompanied by sustainable funding for the product with appropriate training of healthcare staff, and robust quality control and assurance processes. In the absence of quantitative PoC G6PD tests, G6PD status can be gauged with qualitative diagnostics, however none of the available tests is currently sensitive enough to guide TQ treatment. TQ introduction will require overcoming additional challenges including the management of severely and intermediately G6PD deficient individuals. Robust strategies are needed to ensure that effective treatment practices can be deployed widely, and these should ensure that the caveats are outweighed by the benefits of radical cure for both the patients and the community. Widespread access to quality controlled G6PD testing will be critical.

BackgroundArtemisinin-based combination therapy (ACT) is recommended for uncomplicated Plasmodium vivax malaria in areas of emerging chloroquine resistance. We undertook a systematic review and individual patient data meta-analysis to compare the efficacies of dihydroartemisinin-piperaquine (DP) and artemether-lumefantrine (AL) with or without primaquine (PQ) on the risk of recurrent P. vivax.Methods and findingsClinical efficacy studies of uncomplicated P. vivax treated with DP or AL and published between January 1, 2000, and January 31, 2018, were identified by conducting a systematic review registered with the International Prospective Register of Systematic Reviews (PROSPERO): CRD42016053310. Investigators of eligible studies were invited to contribute individual patient data that were pooled using standardised methodology. The effect of mg/kg dose of piperaquine/lumefantrine, ACT administered, and PQ on the rate of P. vivax recurrence between days 7 and 42 after starting treatment were investigated by Cox regression analyses according to an a priori analysis plan. Secondary outcomes were the risk of recurrence assessed on days 28 and 63. Nineteen studies enrolling 2,017 patients were included in the analysis. The risk of recurrent P. vivax at day 42 was significantly higher in the 384 patients treated with AL alone (44.0%, 95% confidence interval [CI] 38.7-49.8) compared with the 812 patients treated with DP alone (9.3%, 95% CI 7.1-12.2): adjusted hazard ratio (AHR) 12.63 (95% CI 6.40-24.92), p < 0.001. The rates of recurrence assessed at days 42 and 63 were associated inversely with the dose of piperaquine: AHRs (95% CI) for every 5-mg/kg increase 0.63 (0.48-0.84), p = 0.0013 and 0.83 (0.73-0.94), p = 0.0033, respectively. The dose of lumefantrine was not significantly associated with the rate of recurrence (1.07 for every 5-mg/kg increase, 95% CI 0.99-1.16, p = 0.0869). In a post hoc analysis, in patients with symptomatic recurrence after AL, the mean haemoglobin increased 0.13 g/dL (95% CI 0.01-0.26) for every 5 days that recurrence was delayed, p = 0.0407. Coadministration of PQ reduced substantially the rate of recurrence assessed at day 42 after AL (AHR = 0.20, 95% CI 0.10-0.41, p < 0.001) and at day 63 after DP (AHR = 0.08, 95% CI 0.01-0.70, p = 0.0233). Results were limited by follow-up of patients to 63 days or less and nonrandomised treatment groups.ConclusionsIn this study, we observed the risk of P. vivax recurrence at day 42 to be significantly lower following treatment with DP compared with AL, reflecting the longer period of post-treatment prophylaxis; this risk was reduced substantially by coadministration with PQ. We found that delaying P. vivax recurrence was associated with a small but significant improvement in haemoglobin. These results highlight the benefits of PQ radical cure and also the provision of blood-stage antimalarial agents with prolonged post-treatment prophylaxis.

IntroductionThe evidence for whether weight loss following longer-term lifestyle interventions results in improved health-related quality of life (HRQoL) is inconclusive. This study examines whether women who lose weight after completing an 18-month web-based lifestyle modification intervention would report a corresponding improvement in HRQoL as measured using the Patient-Reported Outcomes Measurement Information System 29-item profile (PROMIS-29 v1.0).MethodsData from 216 rural women, ages 40 to 69, with baseline and 18-month PROMIS-29 data were analyzed in this secondary analysis of the Women Weigh-in for Wellness clinical trial. This trial promoted lifestyle modification for initial weight loss (baseline to 6 months) and guided weight loss (6 months to 18 months) using a web-delivery format.ResultsAfter adjusting for age, number of comorbidities, change in physical activity from baseline, intervention group, and baseline PROMIS-29 scores, change in weight was associated with improved health-related quality of life (HRQoL) in the domains of depression, physical function, pain interference, fatigue, and satisfaction with social role. Logistic regressions, adjusting for the same factors, indicated women with ≥ 10% weight loss were more likely to report lower depression, higher physical function and less pain interference, compared to women who gained weight or lost < 5%.ConclusionsAmong our rural women, a loss in weight from baseline appeared to be associated with overall improvement in multiple PROMIS-29 v 1.0 domains, noting the likelihood of achieving improvement was significantly higher among women who attained ≥ 10% weight loss. These findings may positively influence a woman's adherence to lifestyle modification weight loss and weight maintenance program.Trial registrationClinicalTrials.gov NCT01307644.

Objective. Purely web-based weight loss and weight-loss maintenance interventions show promise to influence behavior change. Yet, little is known about user engagement with features of web-based interventions that predict clinically meaningful weight loss (≥5% bodyweight loss). This study examines level of website feature engagement with the likelihood of attaining ≥5% bodyweight loss after 6 and 18 months participation in a web-based intervention, among rural women at high risk of obesity-related diseases and disability. Methods. In this secondary analysis of clinical trial data of 201 rural women, we examined weight change and user engagement, measured as clicks on specific web-based intervention features (messaging and self-tracking), as associated with clinically meaningful weight loss (baseline to 6 months) and weight-loss maintenance (6 to 18 months). Results. Generalized estimating equations, adjusted for age, intervention group, and intervention phase, revealed high engagement with messaging predicted whether women achieved ≥5% weight loss at 6 months and at 18 months. There was no effect of self-tracking. Conclusions. Being engaged with messages was associated with attaining clinically meaningful short-term and longer-term weight loss. This trial is registered with NCT01307644.