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Significance Extracellular vesicle (EV)-mediated transfer of macromolecules may play a key role in cellular communication and may have utility in directed molecular therapies. In addition, the EV packaged biomolecules in serum may have potential for diagnosing cancer and determining its likelihood of metastasis. EVs are heterogeneous and there are many outstanding questions associated with biogenesis, uptake, and the fate of transferred molecules in recipient cells. In fact, the function, characterization, and even the nomenclature of EVs are being refined. Here we aimed to improve the functional characterization of EVs, and observed that only microvesicles (MVs), but not exosomes, can functionally transfer loaded reporter molecules to recipient cells, largely by delivering plasmid DNA. Our data show that exosomes and MVs are structurally and functionally distinct. Extracellular vesicles (EVs), specifically exosomes and microvesicles (MVs), are presumed to play key roles in cell–cell communication via transfer of biomolecules between cells. The biogenesis of these two types of EVs differs as they originate from either the endosomal (exosomes) or plasma (MVs) membranes. To elucidate the primary means through which EVs mediate intercellular communication, we characterized their ability to encapsulate and deliver different types of macromolecules from transiently transfected cells. Both EV types encapsulated reporter proteins and mRNA but only MVs transferred the reporter function to recipient cells. De novo reporter protein expression in recipient cells resulted only from plasmid DNA (pDNA) after delivery via MVs. Reporter mRNA was delivered to recipient cells by both EV types, but was rapidly degraded without being translated. MVs also mediated delivery of functional pDNA encoding Cre recombinase in vivo to tissues in transgenic Cre-lox reporter mice. Within the parameters of this study, MVs delivered functional pDNA, but not RNA, whereas exosomes from the same source did not deliver functional nucleic acids. These results have significant implications for understanding the role of EVs in cellular communication and for development of EVs as delivery tools. Moreover, studies using EVs from transiently transfected cells may be confounded by a predominance of pDNA transfer.

Genomic epidemiology uses pathogens’ whole-genome sequences to understand and manage the spread of infectious diseases. Whole-genome data can be used to monitor outbreaks and cluster formation, identify cross-community transmissions, and characterize drug resistance and immune evasion. Typically, bacteria are cultured from clinical samples to obtain DNA for sequencing to generate whole-genome data. However, culture-independent diagnostic methods are utilized for some fastidious bacteria for better diagnostic yield and rapid pathogen genomics. Whole-genome enrichment (WGE) using targeted DNA sequencing enables direct sequencing of clinical samples without having to culture pathogens. However, the cost of capture probes (“baits”) limits the utility of this method for large-scale genomic epidemiology. We developed a cost-effective method named C ircular N ucleic acid E nrichment R eagent s ynthesis (CNERs) to generate whole-genome enrichment probes. We demonstrated the method by producing probes for Mycobacterium tuberculosis , which we used to enrich M. tuberculosis DNA that had been spiked at concentrations as low as 0.01% and 100 genome copies against a human DNA background to 1,225-fold and 4,636-fold. Furthermore, we enriched DNA from different M. tuberculosis lineages and M. bovis and demonstrated the utility of the WGE-CNERs data for lineage identification and drug-resistance characterization using an established pipeline. The CNERs method for whole-genome enrichment will be a valuable tool for the genomic epidemiology of emerging and difficult-to-grow pathogens. Emerging infectious diseases require continuous pathogen monitoring. Rapid clinical diagnosis by nucleic acid amplification is limited to a small number of targets and may miss target detection due to new mutations in clinical isolates. Whole-genome sequencing (WGS) identifies genome-wide variations that may be used to determine a pathogen’s drug resistance patterns and phylogenetically characterize isolates to track disease origin and transmission. WGS is typically performed using DNA isolated from cultured clinical isolates. Culturing clinical specimens increases turn-around time and may not be possible for fastidious bacteria. To overcome some of these limitations, direct sequencing of clinical specimens has been attempted using expensive capture probes to enrich the entire genomes of target pathogens. We present a method to produce a cost-effective, time-efficient, and large-scale synthesis of probes for whole-genome enrichment. We envision that our method can be used for direct clinical sequencing of a wide range of microbial pathogens for genomic epidemiology.

The Escherichia coli ChrR enzyme is an obligatory two-electron quinone reductase that has many applications, such as in chromate bioremediation. Its crystal structure, solved at 2.2 Å resolution, shows that it belongs to the flavodoxin superfamily in which flavin mononucleotide (FMN) is firmly anchored to the protein. ChrR crystallized as a tetramer, and size exclusion chromatography showed that this is the oligomeric form that catalyzes chromate reduction. Within the tetramer, the dimers interact by a pair of two hydrogen bond networks, each involving Tyr128 and Glu146 of one dimer and Arg125 and Tyr85 of the other; the latter extends to one of the redox FMN cofactors. Changes in each of these amino acids enhanced chromate reductase activity of the enzyme, showing that this network is centrally involved in chromate reduction.

Risk assessments and intervention trials have been used by the U.S. Environmental Protection Agency to estimate drinking water health risks. Seldom are both methods used concurrently. Between 2001 and 2003, illness data from a trial were collected simultaneously with exposure data, providing a unique opportunity to compare direct risk estimates of waterborne disease from the intervention trial with indirect estimates from a risk assessment. Comparing the group with water treatment (active) with that without water treatment (sham), the estimated annual attributable disease rate (cases per 10,000 persons per year) from the trial provided no evidence of a significantly elevated drinking water risk [attributable risk = -365 cases/year, sham minus active; 95% confidence interval (CI), -2,555 to 1,825]. The predicted mean rate of disease per 10,000 persons per person-year from the risk assessment was 13.9 (2.5, 97.5 percentiles: 1.6, 37.7) assuming 4 log removal due to viral disinfection and 5.5 (2.5, 97.5 percentiles: 1.4, 19.2) assuming 6 log removal. Risk assessments are important under conditions of low risk when estimates are difficult to attain from trials. In particular, this assessment pointed toward the importance of attaining site-specific treatment data and the clear need for a better understanding of viral removal by disinfection. Trials provide direct risk estimates, and the upper confidence limit estimates, even if not statistically significant, are informative about possible upper estimates of likely risk. These differences suggest that conclusions about waterborne disease risk may be strengthened by the joint use of these two approaches.

Despite preventive vaccines for oncogenic human papillomaviruses (HPVs), cervical intraepithelial neoplasia (CIN) is common, and current treatments are ablative and can lead to long-term reproductive morbidity. We assessed whether VGX-3100, synthetic plasmids targeting HPV-16 and HPV-18 E6 and E7 proteins, delivered by electroporation, would cause histopathological regression in women with CIN2/3. Efficacy, safety, and immunogenicity of VGX-3100 were assessed in CIN2/3 associated with HPV-16 and HPV-18, in a randomised, double-blind, placebo-controlled phase 2b study. Patients from 36 academic and private gynaecology practices in seven countries were randomised (3:1) to receive 6 mg VGX-3100 or placebo (1 mL), given intramuscularly at 0, 4, and 12 weeks. Randomisation was stratified by age (<25 vs ≥25 years) and CIN2 versus CIN3 by computer-generated allocation sequence (block size 4). Funder and site personnel, participants, and pathologists were masked to treatment. The primary efficacy endpoint was regression to CIN1 or normal pathology 36 weeks after the first dose. Per-protocol and modified intention-to-treat analyses were based on patients receiving three doses without protocol violations, and on patients receiving at least one dose, respectively. The safety population included all patients who received at least one dose. The trial is registered at ClinicalTrials.gov (number NCT01304524) and EudraCT (number 2012-001334-33). Between Oct 19, 2011, and July 30, 2013, 167 patients received either VGX-3100 (n=125) or placebo (n=42). In the per-protocol analysis 53 (49·5%) of 107 VGX-3100 recipients and 11 (30·6%) of 36 placebo recipients had histopathological regression (percentage point difference 19·0 [95% CI 1·4–36·6]; p=0·034). In the modified intention-to-treat analysis 55 (48·2%) of 114 VGX-3100 recipients and 12 (30·0%) of 40 placebo recipients had histopathological regression (percentage point difference 18·2 [95% CI 1·3–34·4]; p=0·034). Injection-site reactions occurred in most patients, but only erythema was significantly more common in the VGX-3100 group (98/125, 78·4%) than in the placebo group (24/42, 57·1%; percentage point difference 21·3 [95% CI 5·3–37·8]; p=0·007). VGX-3100 is the first therapeutic vaccine to show efficacy against CIN2/3 associated with HPV-16 and HPV-18. VGX-3100 could present a non-surgical therapeutic option for CIN2/3, changing the treatment outlook for this common disease. Inovio Pharmaceuticals.

A substantial increase in grain yield potential is required, along with better use of water and fertilizer, to ensure food security and environmental protection in future decades. For improvements in photosynthetic capacity to result in additional wheat yield, extra assimilates must be partitioned to developing spikes and grains and/or potential grain weight increased to accommodate the extra assimilates. At the same time, improvement in dry matter partitioning to spikes should ensure that it does not increase stem or root lodging. It is therefore crucial that improvements in structural and reproductive aspects of growth accompany increases in photosynthesis to enhance the net agronomic benefits of genetic modifications. In this article, six complementary approaches are proposed, namely: (i) optimizing developmental pattern to maximize spike fertility and grain number, (ii) optimizing spike growth to maximize grain number and dry matter harvest index, (iii) improving spike fertility through desensitizing floret abortion to environmental cues, (iv) improving potential grain size and grain filling, and (v) improving lodging resistance. Since many of the traits tackled in these approaches interact strongly, an integrative modelling approach is also proposed, to (vi) identify any trade-offs between key traits, hence to define target ideotypes in quantitative terms. The potential for genetic dissection of key traits via quantitative trait loci analysis is discussed for the efficient deployment of existing variation in breeding programmes. These proposals should maximize returns in food production from investments in increased crop biomass by increasing spike fertility, grain number per unit area and harvest index whilst optimizing the trade-offs with potential grain weight and lodging resistance.

Nonlive vaccine approaches that are simple to deliver and stable at room temperature or 2-8°C could be advantageous in controlling future Ebola virus (EBOV) outbreaks. Using an immunopotent DNA vaccine that generates protection from lethal EBOV challenge in small animals and nonhuman primates, we performed a clinical study to evaluate both intramuscular (IM) and novel intradermal (ID) DNA delivery. Two DNA vaccine candidates (INO-4201 and INO-4202) targeting the EBOV glycoprotein (GP) were evaluated for safety, tolerability, and immunogenicity in a phase 1 clinical trial. The candidates were evaluated alone, together, or in combination with plasmid-encoded human cytokine interleukin-12 followed by in vivo electroporation using either the CELLECTRA® IM or ID delivery devices. The safety profile of all 5 regimens was shown to be benign, with the ID route being better tolerated. Antibodies to EBOV GP were generated by all 5 regimens with the fastest and steepest rise observed in the ID group. Cellular immune responses were generated with every regimen. ID delivery of INO-4201 was well tolerated and resulted in 100% seroreactivity after 2 doses and elicited interferon-γ T-cell responses in over 70% of subjects, providing a new approach for EBOV prevention in diverse populations. Clinical Trials Registration. NCT02464670.

Motor cortex (M1) has been thought to form a continuous somatotopic homunculus extending down the precentral gyrus from foot to face representations 1 , 2 , despite evidence for concentric functional zones 3 and maps of complex actions 4 . Here, using precision functional magnetic resonance imaging (fMRI) methods, we find that the classic homunculus is interrupted by regions with distinct connectivity, structure and function, alternating with effector-specific (foot, hand and mouth) areas. These inter-effector regions exhibit decreased cortical thickness and strong functional connectivity to each other, as well as to the cingulo-opercular network (CON), critical for action 5 and physiological control 6 , arousal 7 , errors 8 and pain 9 . This interdigitation of action control-linked and motor effector regions was verified in the three largest fMRI datasets. Macaque and pediatric (newborn, infant and child) precision fMRI suggested cross-species homologues and developmental precursors of the inter-effector system. A battery of motor and action fMRI tasks documented concentric effector somatotopies, separated by the CON-linked inter-effector regions. The inter-effectors lacked movement specificity and co-activated during action planning (coordination of hands and feet) and axial body movement (such as of the abdomen or eyebrows). These results, together with previous studies demonstrating stimulation-evoked complex actions 4 and connectivity to internal organs 10 such as the adrenal medulla, suggest that M1 is punctuated by a system for whole-body action planning, the somato-cognitive action network (SCAN). In M1, two parallel systems intertwine, forming an integrate–isolate pattern: effector-specific regions (foot, hand and mouth) for isolating fine motor control and the SCAN for integrating goals, physiology and body movement. Functional MRI studies across ages show that the classic homunculus of the motor cortex in humans is in fact discontinuous, alternating with action control-linked regions termed the somato-cognitive action network.

Recurrent respiratory papillomatosis (RRP) is a chronic airway disease caused by Human Papillomavirus (HPV). INO-3107, DNA immunotherapy designed to elicit T-cells against HPV-6 and HPV-11, was evaluated in a 52-week Phase 1/2 study for efficacy, safety, and immunogenicity (NCT04398433). Thirty-two eligible adults with HPV-6 and/or HPV-11 RRP, requiring ≥2 surgical interventions in the year preceding dosing were enrolled between October 2020 and November 2021 and administered 4 INO-3107 doses by intramuscular injection followed by electroporation. The primary endpoint was safety and tolerability, as assessed by treatment-emergent adverse events (TEAEs). Secondary endpoints included surgical intervention frequency and change in RRP Severity Score (modified) post-INO-3107 and assessment of immune responses. 81% (26/32) of patients experienced surgery reduction following INO-3107 compared with the year prior to treatment. Blood assessments revealed HPV-6 and HPV-11 antigen-specific T-cell induction. RNA sequencing identified an inflammatory response in papillomas, inclusive of cytolytic CD8 + T-cell signatures. T-cell receptor sequencing revealed emergent T-cell clones in blood and confirmed trafficking to papillomas. Treatment-related adverse events (AEs) were reported in 13/32 (41%) patients, all low-grade. INO-3107 provides clinical benefit to HPV-6 and/or HPV-11-associated RRP adults and is well-tolerated. Importantly, treatment-induced peripheral T-cell responses traffic to airway tissue and are associated with clinical response. In this phase 1/2 trial, the authors show that INO-3107, a DNA immunotherapy designed to elicit an immune response against HPV-6 and -11 recurrent respiratory papillomatosis, is well-tolerated and demonstrates an antigen specific immune response resulting in surgical reduction in 81% of trial participants.