HIV-1 env gene mutations outside the targeting probe affects IPDA efficiency.

The intact proviral DNA assay (IPDA) based on droplet digital PCR was developed to identify intact proviral DNA and quantify HIV-1 latency reservoirs in patients infected with HIV-1. However, the genetic characteristics of different HIV-1 subtypes are non-consistent due to their high mutation and recombination rates. Here, we identified that the IPDA based on the sequences features of an HIV-1 subtype could not effectively detect different HIV-1 subtypes due to the high diversity of HIV-1. Furthermore, we demonstrated that mutations in env gene outside the probe binding site affect the detection efficiency of IPDA. Since mutations in env gene outside the probe binding site may also lead to the formation of stop codons, thereby preventing the formation of viruses and ultimately overestimating the number of HIV-1 latency reservoirs, it is important to address the effect of mutations on the IPDA.

Akaluc bioluminescence offers superior sensitivity to track in vivo dynamics of SARS-CoV-2 infection.

Monitoring in vivo viral dynamics can improve our understanding of pathogenicity and tissue tropism. Because the gene size of RNA viruses is typically small, NanoLuc is the primary choice for accommodation within viral genome. However, NanoLuc/Furimazine and also the conventional firefly luciferase/D-luciferin are known to exhibit relatively low tissue permeability and thus less sensitivity for visualization of deep tissue including lungs. Here, we demonstrated in vivo sufficient visualization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection using the pair of a codon-optimized Akaluc and AkaLumine. We engineered the codon-optimized Akaluc gene possessing the similar GC ratio of SARS-CoV-2. Using the SARS-CoV-2 recombinants carrying the codon-optimized Akaluc, we visualized in vivo infection of respiratory organs, including the tissue-specific differences associated with particular variants. Additionally, we could evaluate the efficacy of antivirals by monitoring changes in Akaluc signals. Overall, we offer an effective technology for monitoring viral dynamics in live animals.

Integrity assay for messenger RNA in mouse and human brain samples and synaptosomal preparations.

Traditionally, RNA integrity evaluation is based on ribosomal RNAs (rRNAs). Nevertheless, gene expression studies are usually focused on protein-coding messenger RNAs (mRNAs). Here, we present an RT-qPCR-based assay, which estimates mRNA integrity by comparing the abundance of 3' and 5' mRNA fragments. The assay was validated using plasmids with cloned 3'- and 5'-ends of the cDNA reflecting different ratios of 3' and 5' cDNA amplicons in partially degraded RNA samples. The accuracy of integrity value was ensured by including primer efficiency. We used 5':3' assay to quantify RNA degradation in heat- and enzyme-degraded mouse and human brain tissue RNA as well as in clinical human brain RNA samples. In addition, the 5':3' assay was suitable for assessing mRNA integrity in synaptosomal preparations that lack rRNAs. We concluded that the 5':3' assay can be used as a reliable method to evaluate mRNA integrity in tissue and subcellular preparations.

Activation of ductal progenitor-like cells from adult human pancreas requires extracellular matrix protein signaling.

Ductal progenitor-like cells are a sub-population of ductal cells in the adult human pancreas that have the potential to contribute to regenerative medicine. However, the microenvironmental cues that regulate their activation are poorly understood. Here, we establish a 3-dimensional suspension culture system containing six defined soluble factors in which primary human ductal progenitor-like and ductal non-progenitor cells survive but do not proliferate. Expansion and polarization occur when suspension cells are provided with a low concentration (5% v/v) of Matrigel, a sarcoma cell product enriched in many extracellular matrix (ECM) proteins. Screening of ECM proteins identified that collagen IV can partially recapitulate the effects of Matrigel. Inhibition of integrin α1ß1, a major collagen IV receptor, negates collagen IV- and Matrigel-stimulated effects. These results demonstrate that collagen IV is a key ECM protein that stimulates the expansion and polarization of human ductal progenitor-like and ductal non-progenitor cells via integrin α1ß1 receptor signaling.

Robust induction of functional astrocytes using NGN2 expression in human pluripotent stem cells.

Emerging evidence of species divergent features of astrocytes coupled with the relative inaccessibility of human brain tissue underscore the utility of human pluripotent stem cell (hPSC) technologies for the generation and study of human astrocytes. However, existing approaches for hPSC-astrocyte generation are typically lengthy or require intermediate purification steps. Here, we establish a rapid and highly scalable method for generating functional human induced astrocytes (hiAs). These hiAs express canonical astrocyte markers, respond to pro-inflammatory stimuli, exhibit ATP-induced calcium transients and support neuronal network development. Moreover, single-cell transcriptomic analyses reveal the generation of highly reproducible cell populations across individual donors, mostly resembling human fetal astrocytes. Finally, hiAs generated from a trisomy 21 disease model identify expected alterations in cell-cell adhesion and synaptic signaling, supporting their utility for disease modeling applications. Thus, hiAs provide a valuable and practical resource for the study of basic human astrocyte function and dysfunction in disease.

Cell culture systems for isolation of SARS-CoV-2 clinical isolates and generation of recombinant virus.

A simple and robust cell culture system is essential for generating authentic SARS-CoV-2 stocks for evaluation of viral pathogenicity, screening of antiviral compounds, and preparation of inactivated vaccines. Evidence suggests that Vero E6, a cell line commonly used in the field to grow SARS-CoV-2, does not support efficient propagation of new viral variants and triggers rapid cell culture adaptation of the virus. We generated a panel of 17 human cell lines overexpressing SARS-CoV-2 entry factors and tested their ability to support viral infection. Two cell lines, Caco-2/AT and HuH-6/AT, demonstrated exceptional susceptibility, yielding highly concentrated virus stocks. Notably, these cell lines were more sensitive than Vero E6 cells in recovering SARS-CoV-2 from clinical specimens. Further, Caco-2/AT cells provided a robust platform for producing genetically reliable recombinant SARS-CoV-2 through a reverse genetics system. These cellular models are a valuable tool for the study of SARS-CoV-2 and its continuously emerging variants.

Antibody-based sex determination of human skeletal remains.

Assignment of biological sex to skeletal remains is critical in the accurate reconstruction of the past. Analysis of sex-chromosome encoded AMELX and AMELY peptides from the enamel protein amelogenin underpins a minimally destructive mass spectrometry (MS) method for sex determination of human remains. However, access to such specialist approaches limits applicability. As a convenient alternative, we generated antibodies that distinguish human AMELX and AMELY. Purified antibodies demonstrated high selectivity and quantitative detection against synthetic peptides by ELISA. Using acid etches of enamel from post-medieval skeletons, antibody determinations corrected osteological uncertainties and matched parallel MS, and for Bronze Age samples where only enamel was preserved, also matched MS analyses. Toward improved throughput, automated stations were applied to analyze 19th-century teeth where sex of individuals was documented, confirming MS can be bypassed. Our immunological tools should underpin development of routine, economical, high-throughput methods for sex determination, potentially even in a field setting.

Top-down reconfiguration of SMA cortical connectivity during action preparation.

The Bereitschaftspotential (BP), a scalp potential recorded in humans during action preparation, is characterized by a slow amplitude increase over fronto-central regions as action execution approaches. We recorded TMS evoked-potentials (TEP) stimulating the supplementary motor area (SMA) at different time-points during a Go/No-Go task to assess whether and how cortical excitability and connectivity of this region change as the BP increases. When approaching BP peak, left SMA reactivity resulted greater. Concurrently, its effective connectivity increased with the left occipital areas, while it decreased with the right inferior frontal gyrus, indicating a fast reconfiguration of cortical networks during the preparation of the forthcoming action. Functional connectivity patterns supported these findings, suggesting a critical role of frequency-specific inter-areal interactions in implementing top-down mechanisms in the sensorimotor system prior to action. These findings reveal that BP time-course reflects quantitative and qualitative changes in SMA communication patterns that shape mechanisms involved in motor readiness.

GATA1 deletion in human pluripotent stem cells increases differentiation yield and maturity of neutrophils.

Human pluripotent stem cell (hPSC)-derived tissues can be used to model diseases in cell types that are challenging to harvest and study at-scale, such as neutrophils. Neutrophil dysregulation, specifically neutrophil extracellular trap (NET) formation, plays a critical role in the prognosis and progression of multiple diseases, including COVID-19. While hPSCs can generate limitless neutrophils (iNeutrophils) to study these processes, current differentiation protocols generate heterogeneous cultures of granulocytes and precursors. Here, we describe a method to improve iNeutrophil differentiations through the deletion of GATA1. GATA1 knockout (KO) iNeutrophils are nearly identical to primary neutrophils in form and function. Unlike wild-type iNeutrophils, GATA1 KO iNeutrophils generate NETs in response to the physiologic stimulant lipopolysaccharide, suggesting they are a more accurate model when performing NET inhibitor screens. Furthermore, through deletion of CYBB, we demonstrate that GATA1 KO iNeutrophils are a powerful tool in determining involvement of a given protein in NET formation.

A quantitative micro-tomographic gut atlas of the lepidopteran model insect Manduca sexta.

The tobacco hornworm is used extensively as a model system for ecotoxicology, immunology and gut physiology. Here, we established a micro-computed tomography approach based on the oral application of the clinical contrast agent iodixanol, allowing for a high-resolution quantitative analysis of the Manduca sexta gut. This technique permitted the identification of previously unknown and understudied structures, such as the crop or gastric ceca, and revealed the underlying complexity of the hindgut folding pattern, which is involved in fecal pellet formation. The acquired data enabled the volume rendering of all gut parts, the reliable calculation of their volumes, and the virtual endoscopy of the entire alimentary tract. It can provide information for accurate orientation in histology uses, enable quantitative anatomical phenotyping in three dimensions, and allow the calculation of locally effective midgut concentrations of applied chemicals. This atlas will provide critical insights into the evolution of the alimentary tract in lepidopterans.

Maximum tree lifespans derived from public-domain dendrochronological data.

The public-domain International Tree-Ring Data Bank (ITRDB) is an under-utilized dataset to improve existing estimates of global tree longevity. We used the longest continuous ring-width series of existing ITRDB collections as an index of maximum tree age for that species and site. Using a total of 3,689 collections, we obtained longevity estimates for 237 unique tree species, 157 conifers and 80 angiosperms, distributed all over the world. More than half of the species (167) were represented by no more than 10 collections, and a similar number of species (144) reached longevity greater than 300 years. Maximum tree ages exceeded 1,000 years for several species (22), all of them conifers, whereas angiosperm longevity peaked around 500 years. Given the current emphasis on identifying human-induced impacts on global systems, detailed analyses of ITRDB holdings provide one of the most reliable sources of information for tree longevity as an ecological trait.

Effects of lysate/tissue storage at -80°C on subsequently extracted EVs of epithelial ovarian cancer tissue origins.

Small extracellular vesicles (sEVs) and large extracellular vesicles (lEVs), play vital roles in intercellular communication. We optimized a method that extracts EVs from epithelial ovarian cancer (EOC) tissues for the purpose of investigating whether cryopreservation of EOC tissues affects the phenotypes, contents, and biological functions of extracted EVs. EV morphology, the number and size distribution of EVs, and EV-related markers were analyzed. Storage of lysates at -80°C decreased lEV yield and increased sEV yield, whereas storage of tissues at -80°C increased both sEV and lEV yields; neither changed the morphology or particle mass ratio of EVs. The two cryopreservation groups retained over 90% of proteins and 80% of miRNAs detected in the "fresh" group. EVs extracted following lysate/tissue storage at -80°C could also promote angiogenesis and invasive migration ability in human endothelial cells. Cryopreserved EOC tissue may benefit clinical applications for studies of tissue-derived EVs, especially EV proteins-related ones.

Detection and annotation of transposable element insertions and deletions on the human genome using nanopore sequencing.

Repetitive sequences represent about 45% of the human genome. Some are transposable elements (TEs) with the ability to change their position in the genome, creating genetic variability both as insertions or deletions, with potential pathogenic consequences. We used long-read nanopore sequencing to identify TE variants in the genomes of 24 patients with antithrombin deficiency. We identified 7 344 TE insertions and 3 056 TE deletions, 2 926 were not previously described in publicly available databases. The insertions affected 3 955 genes, with 6 insertions located in exons, 3 929 in introns, and 147 in promoters. Potential functional impact was evaluated with gene annotation and enrichment analysis, which suggested a strong relationship with neuron-related functions and autism. We conclude that this study encourages the generation of a complete map of TEs in the human genome, which will be useful for identifying new TEs involved in genetic disorders.

Uniform transgene activation in Tet-On systems depends on sustained rtTA expression.

Application of the tetracycline-inducible gene expression system (Tet-On) in human induced pluripotent stem cells (hiPSCs) has become a fundamental transgenic tool owing to its regulatable gene expression. One of the major hurdles in hiPSC application is non-uniform transgene activation. Here, we report that the supplementation of reverse tetracycline transactivator (rtTA) in polyclonal hiPSCs populations can achieve the uniform transgene activation of Tet-On. Furthermore, the choice of antibiotic selection markers connected by an internal ribosomal entry site (IRES) can influence the expression of upstream transgenes. In particular, expression of the rtTA is more uniform in cell populations when linked to puromycin as compared to neomycin, obviating the need for sub-cloning or supplementation of rtTA. Finally, to expand the range of applications, we adopted our findings to tetracycline-inducible MyoD vector (Tet-MyoD). Our Tet-MyoD promises efficient, robust, and reproducible directed myogenic differentiation of hiPSCs.

Epitranscriptome analysis of NAD-capped RNA by spike-in-based normalization and prediction of chronological age.

Nicotinamide adenine dinucleotide (NAD) can be used as an initiating nucleotide in RNA transcription to produce NAD-capped RNA (NAD-RNA). RNA modification by NAD that links metabolite with expressed transcript is a poorly studied epitranscriptomic modification. Current NAD-RNA profiling methods involve multi-steps of chemo-enzymatic labeling and affinity-based enrichment, thus presenting a critical analytical challenge to remove unwanted variations, particularly batch effects. Here, we propose a computational framework, enONE, to remove unwanted variations. We demonstrate that designed spike-in RNA, together with modular normalization procedures and evaluation metrics, can mitigate technical noise, empowering quantitative and comparative assessment of NAD-RNA across different datasets. Using enONE and a human aging cohort, we reveal age-associated features of NAD-capping and further develop an accurate RNA-based aging clock that combines signatures from both transcriptome and NAD-modified epitranscriptome. enONE facilitates the discovery of NAD-RNA responsive to physiological changes, laying an important foundation for functional investigations into this modification.

Low-temperature resin embedding of the whole brain for various precise structures dissection.

Resin embedding combined with ultra-thin sectioning has been widely used in microscopic and electron imaging to acquire precise structural information of biological tissues. However, the existing embedding method was detrimental to quenchable fluorescent signals of precise structures and pH-insensitive fluorescent dyes. Here, we developed a low-temperature chemical polymerization method named HM20-T to maintain weak signals of various precise structures and to decrease background fluorescence. The fluorescence preservation ratio of green fluorescent protein (GFP) tagged presynaptic elements and tdTomato labeled axons doubled. The HM20-T method was suitable for a variety of fluorescent dyes, such as DyLight 488 conjugated Lycopersicon esculentum lectin. Moreover, the brains also retained immunoreactivity after embedding. In summary, the HM20-T method was suitable for the characterization of multi-color labeled precise structures, which would contribute to the acquisition of complete morphology of various biological tissues and to the investigation of composition and circuit connection in the whole brain.

A rationally designed optochemogenetic switch for activating canonical Wnt signaling.

Accurate spatiotemporal control of multicellular self-organization by various signaling pathways is essential for developmental stages. In particular, evolutionarily conserved Wnt signaling serves as a major morphogenetic switch to determine the anteroposterior axis of the embryo. Here, we developed a genetically encoded optochemogenetic Wnt switch, named optochemoWnt, by coupling a blue light-inducible CRY2olig and rapamycin-inducible LRP6c clustering. The rationally designed optochemoWnt successfully modulated Wnt signaling with AND-gated patterns and demonstrated an improved signal-to-noise ratio (SNR). The dual-triggered switch provides a safeguard to prevent signal leakage resulting from ambient light sources under general laboratory conditions. OptochemoWnt expands the molecular toolbox available for the fields of developmental biology and tissue engineering. In addition, the AND-gated strategy of optochemoWnt may be used for other biomedical applications that integrate user defined switch elements with Boolean logic gates.

A multilocus DNA mini-barcode assay to identify twenty vertebrate wildlife species.

The world faces significant challenges in preserving the diversity of vertebrate species due to wildlife crimes. DNA barcoding, an effective molecular marker for insufficient nuclear DNA, is an authentic and quick identification technique to trace the origin of seized samples in forensic investigations. Here, we present a multiplex assay capable of identifying twenty vertebrate wildlife species utilizing twenty species-specific primers that target short fragments of the mitochondrial Cyt b, COI, 16S rRNA, and 12S rRNA genes. The assay achieved strong species specificity and sensitivity with a detection limit as low as 5 pg of DNA input. Additionally, it effectively discriminated a minor contributor (≥1%) from binary mixtures and successfully identified of noninvasive samples, inhibited DNA samples, artificially degraded DNA samples, and case samples, demonstrating a sensitive, robust, practical and easily interpretable tool in screening, and investigating forensic wildlife crimes.

Optimization of the 16S rRNA sequencing analysis pipeline for studying in vitro communities of gut commensals.

While microbial communities inhabit a wide variety of complex natural environments, in vitro culturing enables highly controlled conditions and high-throughput interrogation for generating mechanistic insights. In vitro assemblies of gut commensals have recently been introduced as models for the intestinal microbiota, which plays fundamental roles in host health. However, a protocol for 16S rRNA sequencing and analysis of in vitro samples that optimizes financial cost, time/effort, and accuracy/reproducibility has yet to be established. Here, we systematically identify protocol elements that have significant impact, introduce bias, and/or can be simplified. Our results indicate that community diversity and composition are generally unaffected by substantial protocol streamlining. Additionally, we demonstrate that a strictly aerobic halophile is an effective spike-in for estimating absolute abundances in communities of anaerobic gut commensals. This time- and money-saving protocol should accelerate discovery by increasing 16S rRNA data reliability and comparability and through the incorporation of absolute abundance estimates.

Visualizing lymphocytic choriomeningitis virus infection in cells and living mice.

Mammarenavirus are a large family of enveloped negative-strand RNA viruses that include several agents responsible for severe hemorrhagic fevers. Until now, no FDA-licensed drug has been admitted for treating an arenavirus infection, and only few effective anti-arenavirus drugs have been tested in vivo. In this work, we designed a recombinant reporter arenavirus lymphocytic choriomeningitis virus that stably expressed nanoluciferase (LCMV-Nluc). The LCMV-Nluc was proved to share similar biological properties with wild-type LCMV and the Nluc intensity reliably reflected viral replication both in vitro and in vivo. Replication of the Nluc-encoding virus in living mice can be visualized by real-time bioluminescent imaging, and bioluminescence can be detected in a variety of organs of infected mice. This work provides a novel approach that enables real-time study of the arenavirus infection and is a convenient and valuable tool for screening of compounds that are active against arenaviruses in vitro and in living mice.