Integrating synthetic biology into wearables could expand opportunities for noninvasive monitoring of physiological status, disease states and exposure to pathogens or toxins. However, the operation ...of synthetic circuits generally requires the presence of living, engineered bacteria, which has limited their application in wearables. Here we report lightweight, flexible substrates and textiles functionalized with freeze-dried, cell-free synthetic circuits, including CRISPR-based tools, that detect metabolites, chemicals and pathogen nucleic acid signatures. The wearable devices are activated upon rehydration from aqueous exposure events and report the presence of specific molecular targets by colorimetric changes or via an optical fiber network that detects fluorescent and luminescent outputs. The detection limits for nucleic acids rival current laboratory methods such as quantitative PCR. We demonstrate the development of a face mask with a lyophilized CRISPR sensor for wearable, noninvasive detection of SARS-CoV-2 at room temperature within 90 min, requiring no user intervention other than the press of a button.
Asymptomatic carriers of Plasmodium parasites hamper malaria control and eradication. Achieving malaria eradication requires ultrasensitive diagnostics for low parasite density infections (<100 ...parasites per microliter blood) that work in resource-limited settings (RLS). Sensitive point-of-care diagnostics are also lacking for nonfalciparum malaria, which is characterized by lower density infections and may require additional therapy for radical cure. Molecular methods, such as PCR, have high sensitivity and specificity, but remain high-complexity technologies impractical for RLS. Here we describe a CRISPR-based diagnostic for ultrasensitive detection and differentiation of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae, using the nucleic acid detection platform SHERLOCK (specific high-sensitivity enzymatic reporter unlocking). We present a streamlined, field-applicable, diagnostic comprised of a 10-min SHERLOCK parasite rapid extraction protocol, followed by SHERLOCK for 60 min for Plasmodium species-specific detection via fluorescent or lateral flow strip readout. We optimized one-pot, lyophilized, isothermal assays with a simplified sample preparation method independent of nucleic acid extraction, and showed that these assays are capable of detection below two parasites per microliter blood, a limit of detection suggested by the World Health Organization. Our P. falciparum and P. vivax assays exhibited 100% sensitivity and specificity on clinical samples (5 P. falciparum and 10 P. vivax samples). This work establishes a field-applicable diagnostic for ultrasensitive detection of asymptomatic carriers as well as a rapid point-of-care clinical diagnostic for nonfalciparum malaria species and low parasite density P. falciparum infections.
An integrated, low-cost, sample-to-answer, CRISPR-based diagnostic detects SARS-CoV-2 and variants from unprocessed saliva.
The COVID-19 pandemic highlights the need for diagnostics that can be ...rapidly adapted and deployed in a variety of settings. Several SARS-CoV-2 variants have shown worrisome effects on vaccine and treatment efficacy, but no current point-of-care (POC) testing modality allows their specific identification. We have developed miSHERLOCK, a low-cost, CRISPR-based POC diagnostic platform that takes unprocessed patient saliva; extracts, purifies, and concentrates viral RNA; performs amplification and detection reactions; and provides fluorescent visual output with only three user actions and 1 hour from sample input to answer out. miSHERLOCK achieves highly sensitive multiplexed detection of SARS-CoV-2 and mutations associated with variants B.1.1.7, B.1.351, and P.1. Our modular system enables easy exchange of assays to address diverse user needs and can be rapidly reconfigured to detect different viruses and variants of concern. An adjunctive smartphone application enables output quantification, automated interpretation, and the possibility of remote, distributed result reporting.
Technologies that define the atomic-level structure of neutralization-sensitive epitopes on viral surface proteins are transforming vaccinology and guiding new vaccine development approaches. ...Previously, iterative rounds of protein engineering were performed to preserve the prefusion conformation of the respiratory syncytial virus (RSV) fusion (F) glycoprotein, resulting in a stabilized subunit vaccine candidate (DS-Cav1), which showed promising results in mice and macaques. Here, phase I human immunogenicity data reveal a more than 10-fold boost in neutralizing activity in serum from antibodies targeting prefusion-specific surfaces of RSV F. These findings represent a clinical proof of concept for structure-based vaccine design, suggest that development of a successful RSV vaccine will be feasible, and portend an era of precision vaccinology.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutations may diminish vaccine-induced protective immune responses, particularly as antibody titers wane over time. Here, we assess the ...effect of SARS-CoV-2 variants B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), B.1.429 (Epsilon), B.1.526 (Iota), and B.1.617.2 (Delta) on binding, neutralizing, and angiotensin-converting enzyme 2 (ACE2)–competing antibodies elicited by the messenger RNA (mRNA) vaccine mRNA-1273 over 7 months. Cross-reactive neutralizing responses were rare after a single dose. At the peak of response to the second vaccine dose, all individuals had responses to all variants. Binding and functional antibodies against variants persisted in most subjects, albeit at low levels, for 6 months after the primary series of the mRNA-1273 vaccine. Across all assays, B.1.351 had the lowest antibody recognition. These data complement ongoing studies to inform the potential need for additional boost vaccinations.
ClpC1 is an emerging new target for the treatment of
infections, and several cyclic peptides (ecumicin, cyclomarin A, and lassomycin) are known to act on this target. This study identified another ...group of peptides, the rufomycins (RUFs), as bactericidal to
through the inhibition of ClpC1 and subsequent modulation of protein degradation of intracellular proteins. Rufomycin I (RUFI) was found to be a potent and selective lead compound for both
(MIC, 0.02 μM) and
(MIC, 0.4 μM). Spontaneously generated mutants resistant to RUFI involved seven unique single nucleotide polymorphism (SNP) mutations at three distinct codons within the
-terminal domain of
(V13, H77, and F80). RUFI also significantly decreased the proteolytic capabilities of the ClpC1/P1/P2 complex to degrade casein, while having no significant effect on the ATPase activity of ClpC1. This represents a marked difference from ecumicin, which inhibits ClpC1 proteolysis but stimulates the ATPase activity, thereby providing evidence that although these peptides share ClpC1 as a macromolecular target, their downstream effects are distinct, likely due to differences in binding.
Currently, licensed seasonal influenza vaccines display variable vaccine effectiveness, and there remains a need for novel vaccine platforms capable of inducing broader responses against viral ...protein domains conserved among influenza subtypes. We conducted a first-in-human, randomized, open-label, phase 1 clinical trial ( NCT03186781 ) to evaluate a novel ferritin (H2HA-Ferritin) nanoparticle influenza vaccine platform. The H2 subtype has not circulated in humans since 1968. Adults born after 1968 have been exposed to only the H1 subtype of group 1 influenza viruses, which shares a conserved stem with H2. Including both H2-naive and H2-exposed adults in the trial allowed us to evaluate memory responses against the conserved stem domain in the presence or absence of pre-existing responses against the immunodominant HA head domain. Fifty healthy participants 18-70 years of age received H2HA-Ferritin intramuscularly as a single 20-μg dose (n = 5) or a 60-μg dose either twice in a homologous (n = 25) prime-boost regimen or once in a heterologous (n = 20) prime-boost regimen after a matched H2 DNA vaccine prime. The primary objective of this trial was to evaluate the safety and tolerability of H2HA-Ferritin either alone or in prime-boost regimens. The secondary objective was to evaluate antibody responses after vaccination. Both vaccines were safe and well tolerated, with the most common solicited symptom being mild headache after both H2HA-Ferritin (n = 15, 22%) and H2 DNA (n = 5, 25%). Exploratory analyses identified neutralizing antibody responses elicited by the H2HA-Ferritin vaccine in both H2-naive and H2-exposed populations. Furthermore, broadly neutralizing antibody responses against group 1 influenza viruses, including both seasonal H1 and avian H5 subtypes, were induced in the H2-naive population through targeting the HA stem. This ferritin nanoparticle vaccine technology represents a novel, safe and immunogenic platform with potential application for pandemic preparedness and universal influenza vaccine development.
Acetylation is a protein post-translational modification (PTM) that can affect a variety of cellular processes. In bacteria, two PTM
ε-acetylation mechanisms have been identified: ...non-enzymatic/chemical acetylation via acetyl phosphate or acetyl coenzyme A and enzymatic acetylation via protein acetyltransferases. Prior studies have shown that extensive acetylation of
ε-lysine residues of numerous proteins from a variety of bacteria occurs via non-enzymatic acetylation. In
, new
ε-lysine acetyltransferases (KATs) that enzymatically acetylate other proteins have been identified, thus expanding the repertoire of protein substrates that are potentially regulated by acetylation. Therefore, we designed a study to leverage the wealth of structural data in the Protein Data Bank (PDB) to determine: (1) the 3D location of lysine residues on substrate proteins that are acetylated by
KATs, and (2) investigate whether these residues are conserved on 3D structures of their homologs. Five
KAT substrate proteins that were previously identified as being acetylated by YiaC and had 3D structures in the PDB were selected for further analysis: adenylate kinase (Adk), isocitrate dehydrogenase (Icd), catalase HPII (KatE), methionyl-tRNA formyltransferase (Fmt), and a peroxide stress resistance protein (YaaA). We methodically compared over 350 protein structures of these
enzymes and their homologs; to accurately determine lysine residue conservation requires a strategy that incorporates both flexible structural alignments and visual inspection. Moreover, our results revealed discrepancies in conclusions about lysine residue conservation in homologs when examining linear amino acid sequences compared to 3D structures.
Summary
Objective
To analyse gender differences in the clinical presentation and recovery of paediatric patients with Cushing's disease (CD) after transsphenoidal surgery (TSS). Indeed, gender ...differences between paediatric patients with CD during presentation, after TSS and postoperative recovery have not been adequately studied.
Design
Data were obtained and retrospectively analysed from clinical reports and biochemical tests at the time of presentation, 5–9 days after TSS and at the 6 and 12 months postoperative follow‐up visits to determine hypothalamic–pituitary–adrenal axis (HPAA) recovery.
Patients
Data from 102 paediatric patients (48 females, 54 males, mean age 12·9 ± 3·0) with CD who underwent TSS at the National Institute of Health (NIH) Clinical Center between 1997 and 2011.
Results
There was equal distribution of paediatric CD between males and females (53% vs 47%; n = 102, P = 0·484). Males were more likely than females to present with higher mean BMI Z‐scores (2·2 ± 0·7 vs 1·9 ± 0·6, P = 0·0079), lower mean height Z‐scores (−1·2 ± 1·3 vs −0·7 ± 1·1, P = 0·0467) and higher median plasma ACTH (12·2 vs 8·5 pmol/l; P = 0·0495). Females did not present more frequently with any single sign or symptom. No significant differences were found between males and females for CD cure rates 5–9 days after TSS (87·0% males vs 87·5% females, P = 1·0), long‐term cure rates (86·5% vs 93·7%; n = 69; P = 0·4374) and HPAA recovery time (11·2 ± 2·5 vs 11·7 ± 2·5 months; n = 47; P = 0·1992).
Conclusions
Paediatric CD is found to have equal distribution between males and females, but male patients present with elevated BMI and potentially shorter height and higher plasma ACTH. There is no significant difference in the cure rate or HPAA recovery time after TSS between males and females.
Adeno-associated viral vector-mediated transfer of DNA coding for broadly neutralizing anti-HIV antibodies (bnAbs) offers an alternative to attempting to induce protection by vaccination or by ...repeated infusions of bnAbs. In this study, we administered a recombinant bicistronic adeno-associated virus (AAV8) vector coding for both the light and heavy chains of the potent broadly neutralizing HIV-1 antibody VRC07 (AAV8-VRC07) to eight adults living with HIV. All participants remained on effective anti-retroviral therapy (viral load (VL) <50 copies per milliliter) throughout this phase 1, dose-escalation clinical trial ( NCT03374202 ). AAV8-VRC07 was given at doses of 5 × 10
, 5 × 10
and 2.5 × 10
vector genomes per kilogram by intramuscular (IM) injection. Primary endpoints of this study were to assess the safety and tolerability of AAV8-VRC07; to determine the pharmacokinetics and immunogenicity of in vivo VRC07 production; and to describe the immune response directed against AAV8-VRC07 vector and its products. Secondary endpoints were to assess the clinical effects of AAV8-VRC07 on CD4 T cell count and VL and to assess the persistence of VRC07 produced in participants. In this cohort, IM injection of AAV8-VRC07 was safe and well tolerated. No clinically significant change in CD4 T cell count or VL occurred during the 1-3 years of follow-up reported here. In participants who received AAV8-VRC07, concentrations of VRC07 were increased 6 weeks (P = 0.008) and 52 weeks (P = 0.016) after IM injection of the product. All eight individuals produced measurable amounts of serum VRC07, with maximal VRC07 concentrations >1 µg ml
in three individuals. In four individuals, VRC07 serum concentrations remained stable near maximal concentration for up to 3 years of follow-up. In exploratory analyses, neutralizing activity of in vivo produced VRC07 was similar to that of in vitro produced VRC07. Three of eight participants showed a non-idiotypic anti-drug antibody (ADA) response directed against the Fab portion of VRC07. This ADA response appeared to decrease the production of serum VRC07 in two of these three participants. These data represent a proof of concept that adeno-associated viral vectors can durably produce biologically active, difficult-to-induce bnAbs in vivo, which could add valuable new tools to the fight against infectious diseases.
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