High-throughput tagging of endogenous loci for rapid characterization of protein function.

To facilitate the interrogation of protein function at scale, we have developed high-throughput insertion of tags across the genome (HITAG). HITAG enables users to rapidly produce libraries of cells, each with a different protein of interest C-terminally tagged. HITAG is based on a modified strategy for performing Cas9-based targeted insertions, coupled with an improved approach for selecting properly tagged lines. Analysis of the resulting clones generated by HITAG reveals high tagging specificity, with most successful tagging events being indel free. Using HITAG, we fuse mCherry to a set of 167 stress granule-associated proteins and elucidate the features that drive a subset of proteins to strongly accumulate within these transient RNA-protein granules.

A dual-luciferase bioluminescence system for the assessment of cellular therapies.

Bioluminescence imaging is a well-established platform for evaluating engineered cell therapies in preclinical studies. However, despite the discovery of new luciferases and substrates, optimal combinations to simultaneously monitor two cell populations remain limited. This makes the functional assessment of cellular therapies cumbersome and expensive, especially in preclinical in vivo models. In this study, we explored the potential of using a green bioluminescence-emitting click beetle luciferase, CBG99, and a red bioluminescence-emitting firefly luciferase mutant, Akaluc, together to simultaneously monitor two cell populations. Using various chimeric antigen receptor T cells and tumor pairings, we demonstrate that these luciferases are suitable for real-time tracking of two cell types using 2D and 3D cultures in vitro and experimental models in vivo. Our data show the broad compatibility of this dual-luciferase (duo-luc) system with multiple bioluminescence detection equipment ranging from benchtop spectrophotometers to live animal imaging systems. Although this study focused on investigating complex CAR T cells and tumor cell interactions, this duo-luc system has potential utility for the simultaneous monitoring of any two cellular components-for example, to unravel the impact of a specific genetic variant on clonal dominance in a mixed population of tumor cells.

Population-specific responses to developmental temperature in the arboviral vector Aedes albopictus: Implications for climate change.

The increase of environmental temperature due to current global warming is not only favouring the expansion of the distribution range of many insect species, but it is also changing their phenology. Insect phenology is tightly linked to developmental timing, which is regulated by environmental temperatures. However, the degree to which the effects of developmental temperatures extend across developmental stages and their inter-stage relationships have not been thoroughly quantified in mosquitoes. Here, we used the mosquito Aedes albopictus, which is an aggressive invasive species and an arboviral vector, to study how developmental temperature influences fitness across developmental stages, thermal traits, energy reserves, transcriptome and Wolbachia prevalence in laboratory-reared populations originally collected from either temperate or tropical regions. We show that hatchability, larval and pupal viability and developmental speed are strongly influenced by temperature, and these effects extend to wing length, body mass, longevity and content of water, protein and lipids in adults in a population-specific manner. On the contrary, neither adult thermal preference nor heat resistance significantly change with temperature. Wolbachia density was generally lower in adult mosquitoes reared at 18°C than at other tested temperatures, and transcriptome analysis showed enrichment for functions linked to stress responses (i.e. cuticle proteins and chitin, cytochrome p450 and heat shock proteins) in mosquitoes reared at both 18 and 32°C. Our data showed an overall reduced vector fitness performance when mosquitoes were reared at 32°C, and the absence of isomorphy in the relationship between developmental stages and temperature in the laboratory population deriving from larvae collected in northern Italy. Altogether, these results have important implications for reliable model projections of the invasion potentials of Ae. albopictus and its epidemiological impact.

The Effect of Ultrasound on the Extraction and Functionality of Proteins from Duckweed (Lemna minor).

The inclusion of protein in the regular human diet is important for the prevention of several chronic diseases. In the search for novel alternative protein sources, plant-based proteins are widely explored from a sustainable and ecological point of view. Duckweed (Lemna minor), also known as water lentil, is an aquatic plant with potential applications for human consumption due to its protein content and carbohydrate contents. Among all the conventional and novel protein extraction methods, the utilization of ultrasound has attracted the attention of scientists because of its effects on improving protein extraction and its functionalities. In this work, a Box-Behnken experimental design was proposed to optimize the alkaline extraction of protein from duckweed. In addition, an exploration of the effects of ultrasound on the morphological, structural, and functional properties of the extracted protein was also addressed. The optimal extraction parameters were a pH of 11.5 and an ultrasound amplitude and processing time of 60% and 20 min, respectively. These process conditions doubled the protein content extracted in comparison to the value from the initial duckweed sample. Furthermore, the application of ultrasound during the extraction of protein generated changes in the FTIR spectra, color, and structure of the duckweed protein, which resulted in improvements in its solubility, emulsifying properties, and foaming capacity.

Targeting IDH2R140Q and other neoantigens in acute myeloid leukemia.

ABSTRACT: For patients with high-risk or relapsed/refractory acute myeloid leukemia (AML), allogeneic stem cell transplantation (allo-HSCT) and the graft-versus-leukemia effect mediated by donor T cells, offer the best chance of long-term remission. However, the concurrent transfer of alloreactive T cells can lead to graft-versus-host disease that is associated with transplant-related morbidity and mortality. Furthermore, ∼60% of patients will ultimately relapse after allo-HSCT, thus, underscoring the need for novel therapeutic strategies that are safe and effective. In this study, we explored the feasibility of immunotherapeutically targeting neoantigens, which arise from recurrent nonsynonymous mutations in AML and thus represent attractive targets because they are exclusively present on the tumor. Focusing on 14 recurrent driver mutations across 8 genes found in AML, we investigated their immunogenicity in 23 individuals with diverse HLA profiles. We demonstrate the immunogenicity of AML neoantigens, with 17 of 23 (74%) reactive donors screened mounting a response. The most immunodominant neoantigens were IDH2R140Q (n = 11 of 17 responders), IDH1R132H (n = 7 of 17), and FLT3D835Y (n = 6 of 17). In-depth studies of IDH2R140Q-specific T cells revealed the presence of reactive CD4+ and CD8+ T cells capable of recognizing distinct mutant-specific epitopes restricted to different HLA alleles. These neo-T cells could selectively recognize and kill HLA-matched AML targets endogenously expressing IDH2R140Q both in vitro and in vivo. Overall, our findings support the clinical translation of neoantigen-specific T cells to treat relapsed/refractory AML.

Targeted DNA integration in human cells without double-strand breaks using CRISPR-associated transposases.

Conventional genome engineering with CRISPR-Cas9 creates double-strand breaks (DSBs) that lead to undesirable byproducts and reduce product purity. Here we report an approach for programmable integration of large DNA sequences in human cells that avoids the generation of DSBs by using Type I-F CRISPR-associated transposases (CASTs). We optimized DNA targeting by the QCascade complex through protein design and developed potent transcriptional activators by exploiting the multi-valent recruitment of the AAA+ ATPase TnsC to genomic sites targeted by QCascade. After initial detection of plasmid-based integration, we screened 15 additional CAST systems from a wide range of bacterial hosts, identified a homolog from Pseudoalteromonas that exhibits improved activity and further increased integration efficiencies. Finally, we discovered that bacterial ClpX enhances genomic integration by multiple orders of magnitude, likely by promoting active disassembly of the post-integration CAST complex, akin to its known role in Mu transposition. Our work highlights the ability to reconstitute complex, multi-component machineries in human cells and establishes a strong foundation to exploit CRISPR-associated transposases for eukaryotic genome engineering.

Molecular mechanisms of SARS-CoV-2 resistance to nirmatrelvir.

Nirmatrelvir is a specific antiviral drug that targets the main protease (Mpro) of SARS-CoV-2 and has been approved to treat COVID-191,2. As an RNA virus characterized by high mutation rates, whether SARS-CoV-2 will develop resistance to nirmatrelvir is a question of concern. Our previous studies have shown that several mutational pathways confer resistance to nirmatrelvir, but some result in a loss of viral replicative fitness, which is then compensated for by additional alterations3. The molecular mechanisms for this observed resistance are unknown. Here we combined biochemical and structural methods to demonstrate that alterations at the substrate-binding pocket of Mpro can allow SARS-CoV-2 to develop resistance to nirmatrelvir in two distinct ways. Comprehensive studies of the structures of 14 Mpro mutants in complex with drugs or substrate revealed that alterations at the S1 and S4 subsites substantially decreased the level of inhibitor binding, whereas alterations at the S2 and S4' subsites unexpectedly increased protease activity. Both mechanisms contributed to nirmatrelvir resistance, with the latter compensating for the loss in enzymatic activity of the former, which in turn accounted for the restoration of viral replicative fitness, as observed previously3. Such a profile was also observed for ensitrelvir, another clinically relevant Mpro inhibitor. These results shed light on the mechanisms by which SARS-CoV-2 evolves to develop resistance to the current generation of protease inhibitors and provide the basis for the design of next-generation Mpro inhibitors.

A continuous process in mixers-settlers for the removal/recovery of chromium in effluents from the tanning industry.

One of the main environmental issues caused by the tanning industry is given by the high concentration of chromium contained on its effluents. The removal of this pollutant has become a technological challenge. To solve this issue, this work proposes a continuous process based on mixers-settlers for the removal of the chromium present in effluents from the tanning industry. The process involves the use of liquid-liquid extraction systems. The study includes the development of isotherms for the removal and stripping, which are further represented through a mathematical model to determine the number of theoretical extraction stages and other operational variables. The results show that a better extraction is achieved in a system with two theoretical stages using Cyanex 272 as extractant, reaching more than 94% of removal of chromium with an extractant concentration of 0.32 mol/L. For stripping, sulfuric acid is used, obtaining a maximum recovery of 94%.

Focused ultrasound-mediated brain genome editing.

Gene editing in the brain has been challenging because of the restricted transport imposed by the blood-brain barrier (BBB). Current approaches mainly rely on local injection to bypass the BBB. However, such administration is highly invasive and not amenable to treating certain delicate regions of the brain. We demonstrate a safe and effective gene editing technique by using focused ultrasound (FUS) to transiently open the BBB for the transport of intravenously delivered CRISPR/Cas9 machinery to the brain.

Rapid and sustained contact tracing training for COVID-19 in San Francisco: a training model for developing an emergency public health workforce.

The City and County of San Francisco was the first municipality in the United States to institute a COVID-19 contact tracing program. The San Francisco Department of Public Health (SFDPH) and the University of California, San Francisco (UCSF) created an outcome-based fully remote contact tracing curriculum using participatory learning methods to train non-public health emergency workers as contact tracers. Between April and December 2020, we trained over 300 individuals in contact tracing skills and procedures over three training phases. Using iterative curriculum design and Kirkpatrick's evaluation methodology, we aimed to ensure high quality and successful person-centered contact tracing. The resulting curriculum consisted of 24 learning outcomes taught with six participatory skills development activities, asynchronous materials, and one-on-one contact tracer support. We collected more than 700 responses from trainees using various evaluation tools across the training phases, and contact tracers interviewed more than 24,000 contacts after training in our program. Our evaluations showed that knowledge and skills improved for most trainees and demonstrated the utility of the training program in preparing trainees to perform person-centered contact tracing in San Francisco. Local health jurisdictions and state health agencies can use this model of curriculum development and evaluation to rapidly train a non-public health workforce to respond to future public health emergencies.

Measuring the Effectiveness of a Multicomponent Program to Manage Academic Stress through a Resilience to Stress Index.

In this work, we evaluate the effectiveness of a multicomponent program that includes psychoeducation in academic stress, mindfulness training, and biofeedback-assisted mindfulness, while enhancing the Resilience to Stress Index (RSI) of students through the control of autonomic recovery from psychological stress. Participants are university students enrolled in a program of excellence and are granted an academic scholarship. The dataset consists of an intentional sample of 38 undergraduate students with high academic performance, 71% (27) women, 29% (11) men, and 0% (0) non-binary, with an average age of 20 years. The group belongs to the "Leaders of Tomorrow" scholarship program from Tecnológico de Monterrey University, in Mexico. The program is structured in 16 individual sessions during an eight-week period, divided into three phases: pre-test evaluation, training program, and post-test evaluation. During the evaluation test, an assessment of the psychophysiological stress profile is performed while the participants undergo a stress test; it includes simultaneous recording of skin conductance, breathing rate, blood volume pulse, heart rate, and heart rate variability. Based on the pre-test and post-test psychophysiological variables, an RSI is computed under the assumption that changes in physiological signals due to stress can be compared against a calibration stage. The results show that approximately 66% of the participants improved their academic stress management after the multicomponent intervention program. A Welch's t-test showed a difference in mean RSI scores (t = -2.30, p = 0.025) between the pre-test and post-test phases. Our findings show that the multicomponent program promoted positive changes in the RSI and in the management of the psychophysiological responses to academic stress.

Targeted DNA integration in human cells without double-strand breaks using CRISPR RNA-guided transposases.

Traditional genome-editing reagents such as CRISPR-Cas9 achieve targeted DNA modification by introducing double-strand breaks (DSBs), thereby stimulating localized DNA repair by endogenous cellular repair factors. While highly effective at generating heterogenous knockout mutations, this approach suffers from undesirable byproducts and an inability to control product purity. Here we develop a system in human cells for programmable, DSB-free DNA integration using Type I CRISPR-associated transposons (CASTs). To adapt our previously described CAST systems, we optimized DNA targeting by the QCascade complex through a comprehensive assessment of protein design, and we developed potent transcriptional activators by exploiting the multi-valent recruitment of the AAA+ ATPase, TnsC, to genomic sites targeted by QCascade. After initial detection of plasmid-based transposition, we screened 15 homologous CAST systems from a wide range of bacterial hosts, identified a CAST homolog from Pseudoalteromonas that exhibited improved activity, and increased integration efficiencies through parameter optimization. We further discovered that bacterial ClpX enhances genomic integration by multiple orders of magnitude, and we propose that this critical accessory factor functions to drive active disassembly of the post-transposition CAST complex, akin to its demonstrated role in Mu transposition. Our work highlights the ability to functionally reconstitute complex, multi-component machineries in human cells, and establishes a strong foundation to realize the full potential of CRISPR-associated transposons for human genome engineering.

Focused ultrasound-mediated brain genome editing.

Gene editing in the mammalian brain has been challenging because of the restricted transport imposed by the blood-brain barrier (BBB). Current approaches rely on local injection to bypass the BBB. However, such administration is highly invasive and not amenable to treating certain delicate regions of the brain. We demonstrate a safe and effective gene editing technique by using focused ultrasound (FUS) to transiently open the BBB for the transport of intravenously delivered CRISPR/Cas9 machinery to the brain.

Development and applications of a CRISPR activation system for facile genetic overexpression in Candida albicans.

For the fungal pathogen Candida albicans, genetic overexpression readily occurs via a diversity of genomic alterations, such as aneuploidy and gain-of-function mutations, with important consequences for host adaptation, virulence, and evolution of antifungal drug resistance. Given the important role of overexpression on C. albicans biology, it is critical to develop and harness tools that enable the analysis of genes expressed at high levels in the fungal cell. Here, we describe the development, optimization, and application of a novel, single-plasmid-based CRISPR activation (CRISPRa) platform for targeted genetic overexpression in C. albicans, which employs a guide RNA to target an activator complex to the promoter region of a gene of interest, thus driving transcriptional expression of that gene. Using this system, we demonstrate the ability of CRISPRa to drive high levels of gene expression in C. albicans, and we assess optimal guide RNA targeting for robust and constitutive overexpression. We further demonstrate the specificity of the system via RNA sequencing. We highlight the application of CRISPR activation to overexpress genes involved in pathogenesis and drug susceptibility, and contribute toward the identification of novel phenotypes. Consequently, this tool will facilitate a broad range of applications for the study of C. albicans genetic overexpression.

Multiple pathways for SARS-CoV-2 resistance to nirmatrelvir.

Nirmatrelvir, an oral antiviral targeting the 3CL protease of SARS-CoV-2, has been demonstrated to be clinically useful against COVID-19 (refs. 1,2). However, because SARS-CoV-2 has evolved to become resistant to other therapeutic modalities3-9, there is a concern that the same could occur for nirmatrelvir. Here we examined this possibility by in vitro passaging of SARS-CoV-2 in nirmatrelvir using two independent approaches, including one on a large scale. Indeed, highly resistant viruses emerged from both and their sequences showed a multitude of 3CL protease mutations. In the experiment peformed with many replicates, 53 independent viral lineages were selected with mutations observed at 23 different residues of the enzyme. Nevertheless, several common mutational pathways to nirmatrelvir resistance were preferred, with a majority of the viruses descending from T21I, P252L or T304I as precursor mutations. Construction and analysis of 13 recombinant SARS-CoV-2 clones showed that these mutations mediated only low-level resistance, whereas greater resistance required accumulation of additional mutations. E166V mutation conferred the strongest resistance (around 100-fold), but this mutation resulted in a loss of viral replicative fitness that was restored by compensatory changes such as L50F and T21I. Our findings indicate that SARS-CoV-2 resistance to nirmatrelvir does readily arise via multiple pathways in vitro, and the specific mutations observed herein form a strong foundation from which to study the mechanism of resistance in detail and to inform the design of next-generation protease inhibitors.

Contamination assessment and potential sources of heavy metals and other elements in sediments of a basin impacted by 500 years of mining in central Mexico.

Since the middle of the 1500 s, mining has been active in central Mexico. Total estimates for low-grade piles and mine tailing materials in the Guanajuato mining district (GMD) are in the range of 150 million tons, covering an area of 15 to 20 km2. GMD is located in the Guanajuato River sub-basin (GRB), which is part of one of the largest basins in Mexico (Lerma-Santiago). Previous studies on the GRB found unusually high concentrations of heavy metals in mining tailings and sediments. Geochemical and statistical methods were used here to determine the sediment's origin, background values, degree of contamination, and toxicity through different contamination indices. This analysis shows that Cu, Co, As, Sb, and Hg are higher than they are in the upper continental crust (UCC) overbank sediments without human and mining influence, because of the ore deposits and rock weathering in GRB. Geochemistry results in stream sediments show anomalies, where Hg, Cu, Zn, As, and Pb are higher than UCC because those heavy metals and trace elements (HMT) have been influenced by human activities and mineral recovery (smelting, amalgamation, cyanidation). The distribution of high concentrations of HMTs and contamination indices occur in the main channel of the Guanajuato River and downstream of the city of Guanajuato. Statistical analyses (cluster and principal component analysis) reveal relationships between Cr, Ni, Cu, and Pb, which are primarily of natural origin, related to rocks of the upper basin. The middle and lower basins are distinctive in their associations between As, Sb, Zn, Pb, and Hg. Additionally, it is recognized that the origins of Pb, Zn, and Hg are geogenic and anthropogenic. This study demonstrates how crucial it is to understand the geochemistry of various HMT sources, with both natural and anthropogenic contributions (stream sediments and rocks), in order to calculate a more realistic background in a basin with both natural anomalies and anthropogenic contamination. The basin is a regional aquifer recharge area, so the new geochemical data are important for improving basin environmental management.

Secreted Fas Decoys Enhance the Antitumor Activity of Engineered and Bystander T Cells in Fas Ligand-Expressing Solid Tumors.

T-cell immunotherapy has demonstrated remarkable clinical outcomes in certain hematologic malignancies. However, efficacy in solid tumors has been suboptimal, partially due to the hostile tumor microenvironment composed of immune-inhibitory molecules. One such suppressive agent abundantly expressed in solid tumors is Fas ligand (FasL), which can trigger apoptosis of Fas-expressing effector cells such as T cells and natural killer (NK) cells. To alleviate this FasL-induced suppression of tumor-specific immune cells in solid tumors, we describe here the development of a Fas decoy that is secreted by engineered cells upon activation and sequesters the ligand, preventing it from engaging with Fas on the surface of effector cells. We further improved the immune-stimulatory effects of this approach by creating a Fas decoy and IL15 cytokine fusion protein, which enhanced the persistence and antitumor activity of decoy-engineered as well as bystander chimeric-antigen receptor (CAR) T cells in xenograft models of pancreatic cancer. Our data indicate that secreted Fas decoys can augment the efficacy of both adoptively transferred and endogenous tumor-specific effector cells in FasL-expressing solid tumors.

Multiple pathways for SARS-CoV-2 resistance to nirmatrelvir.

Nirmatrelvir, an oral antiviral targeting the 3CL protease of SARS-CoV-2, has been demonstrated to be clinically useful in reducing hospitalization or death due to COVID-19 1,2 . However, as SARS-CoV-2 has evolved to become resistant to other therapeutic modalities 3â€"9 , there is a concern that the same could occur for nirmatrelvir. Here, we have examined this possibility by in vitro passaging of SARS-CoV-2 in increasing concentrations of nirmatrelvir using two independent approaches, including one on a large scale in 480 wells. Indeed, highly resistant viruses emerged from both, and their sequences revealed a multitude of 3CL protease mutations. In the experiment done at a larger scale with many replicates, 53 independent viral lineages were selected with mutations observed at 23 different residues of the enzyme. Yet, several common mutational pathways to nirmatrelvir resistance were preferred, with a majority of the viruses descending from T21I, P252L, or T304I as precursor mutations. Construction and analysis of 13 recombinant SARS-CoV-2 clones, each containing a unique mutation or a combination of mutations showed that the above precursor mutations only mediated low-level resistance, whereas greater resistance required accumulation of additional mutations. E166V mutation conferred the strongest resistance (~100-fold), but this mutation resulted in a loss of viral replicative fitness that was restored by compensatory changes such as L50F and T21I. Structural explanations are discussed for some of the mutations that are proximal to the drug-binding site, as well as cross-resistance or lack thereof to ensitrelvir, another clinically important 3CL protease inhibitor. Our findings indicate that SARS-CoV-2 resistance to nirmatrelvir does readily arise via multiple pathways in vitro , and the specific mutations observed herein form a strong foundation from which to study the mechanism of resistance in detail and to inform the design of next generation protease inhibitors.

Functional map of SARS-CoV-2 3CL protease reveals tolerant and immutable sites.

The SARS-CoV-2 3CL protease (3CLpro) is an attractive therapeutic target, as it is essential to the virus and highly conserved among coronaviruses. However, our current understanding of its tolerance to mutations is limited. Here, we develop a yeast-based deep mutational scanning approach to systematically profile the activity of all possible single mutants of the 3CLpro and validate a subset of our results within authentic viruses. We reveal that the 3CLpro is highly malleable and is capable of tolerating mutations throughout the protein. Yet, we also identify specific residues that appear immutable, suggesting that these may be targets for future 3CLpro inhibitors. Finally, we utilize our screening as a basis to identify E166V as a resistance-conferring mutation against the clinically used 3CLpro inhibitor, nirmatrelvir. Collectively, the functional map presented herein may serve as a guide to better understand the biological properties of the 3CLpro and for drug development against coronaviruses.