Biosensors based on peptide exposure show single molecule conformations in live cells.

We describe an approach to study the conformation of individual proteins during single particle tracking (SPT) in living cells. "Binder/tag" is based on incorporation of a 7-mer peptide (the tag) into a protein where its solvent exposure is controlled by protein conformation. Only upon exposure can the peptide specifically interact with a reporter protein (the binder). Thus, simple fluorescence localization reflects protein conformation. Through direct excitation of bright dyes, the trajectory and conformation of individual proteins can be followed. Simple protein engineering provides highly specific biosensors suitable for SPT and FRET. We describe tagSrc, tagFyn, tagSyk, tagFAK, and an orthogonal binder/tag pair. SPT showed slowly diffusing islands of activated Src within Src clusters and dynamics of activation in adhesions. Quantitative analysis and stochastic modeling revealed in vivo Src kinetics. The simplicity of binder/tag can provide access to diverse proteins.

TCR+/BCR+ dual-expressing cells and their associated public BCR clonotype are not enriched in type 1 diabetes.

Ahmed and colleagues recently described a novel hybrid lymphocyte expressing both a B and T cell receptor, termed double expresser (DE) cells. DE cells in blood of type 1 diabetes (T1D) subjects were present at increased numbers and enriched for a public B cell clonotype. Here, we attempted to reproduce these findings. While we could identify DE cells by flow cytometry, we found no association between DE cell frequency and T1D status. We were unable to identify the reported public B cell clone, or any similar clone, in bulk B cells or sorted DE cells from T1D subjects or controls. We also did not observe increased usage of the public clone VH or DH genes in B cells or in sorted DE cells. Taken together, our findings suggest that DE cells and their alleged public clonotype are not enriched in T1D. This Matters Arising paper is in response to Ahmed et al. (2019), published in Cell. See also the response by Ahmed et al. (2021), published in this issue.

Extracellular Vesicle and Particle Biomarkers Define Multiple Human Cancers.

There is an unmet clinical need for improved tissue and liquid biopsy tools for cancer detection. We investigated the proteomic profile of extracellular vesicles and particles (EVPs) in 426 human samples from tissue explants (TEs), plasma, and other bodily fluids. Among traditional exosome markers, CD9, HSPA8, ALIX, and HSP90AB1 represent pan-EVP markers, while ACTB, MSN, and RAP1B are novel pan-EVP markers. To confirm that EVPs are ideal diagnostic tools, we analyzed proteomes of TE- (n = 151) and plasma-derived (n = 120) EVPs. Comparison of TE EVPs identified proteins (e.g., VCAN, TNC, and THBS2) that distinguish tumors from normal tissues with 90% sensitivity/94% specificity. Machine-learning classification of plasma-derived EVP cargo, including immunoglobulins, revealed 95% sensitivity/90% specificity in detecting cancer. Finally, we defined a panel of tumor-type-specific EVP proteins in TEs and plasma, which can classify tumors of unknown primary origin. Thus, EVP proteins can serve as reliable biomarkers for cancer detection and determining cancer type.

The Translational Landscape of the Human Heart.

Gene expression in human tissue has primarily been studied on the transcriptional level, largely neglecting translational regulation. Here, we analyze the translatomes of 80 human hearts to identify new translation events and quantify the effect of translational regulation. We show extensive translational control of cardiac gene expression, which is orchestrated in a process-specific manner. Translation downstream of predicted disease-causing protein-truncating variants appears to be frequent, suggesting inefficient translation termination. We identify hundreds of previously undetected microproteins, expressed from lncRNAs and circRNAs, for which we validate the protein products in vivo. The translation of microproteins is not restricted to the heart and prominent in the translatomes of human kidney and liver. We associate these microproteins with diverse cellular processes and compartments and find that many locate to the mitochondria. Importantly, dozens of microproteins are translated from lncRNAs with well-characterized noncoding functions, indicating previously unrecognized biology.

Multisensory Logic of Infant-Directed Aggression by Males.

Newborn mice emit signals that promote parenting from mothers and fathers but trigger aggressive responses from virgin males. Although pup-directed attacks by males require vomeronasal function, the specific infant cues that elicit this behavior are unknown. We developed a behavioral paradigm based on reconstituted pup cues and showed that discrete infant morphological features combined with salivary chemosignals elicit robust male aggression. Seven vomeronasal receptors were identified based on infant-mediated activity, and the involvement of two receptors, Vmn2r65 and Vmn2r88, in infant-directed aggression was demonstrated by genetic deletion. Using the activation of these receptors as readouts for biochemical fractionation, we isolated two pheromonal compounds, the submandibular gland protein C and hemoglobins. Unexpectedly, none of the identified vomeronasal receptors and associated cues were specific to pups. Thus, infant-mediated aggression by virgin males relies on the recognition of pup's physical traits in addition to parental and infant chemical cues.

Discovery of cytosine deaminases enables base-resolution methylome mapping using a single enzyme.

Deaminases have important uses in modification detection and genome editing. However, the range of applications is limited by the small number of characterized enzymes. To expand the toolkit of deaminases, we developed an in vitro approach that bypasses a major hurdle with their toxicity in cells. We assayed 175 putative cytosine deaminases on a variety of substrates and found a broad range of activity on double- and single-stranded DNA in various sequence contexts, including CpG-specific deaminases and enzymes without sequence preference. We also characterized enzyme selectivity across six DNA modifications and reported enzymes that do not deaminate modified cytosines. The detailed analysis of diverse deaminases opens new avenues for biotechnological and medical applications. As a demonstration, we developed SEM-seq, a non-destructive single-enzyme methylation sequencing method using a modification-sensitive double-stranded DNA deaminase. The streamlined protocol enables accurate, base-resolution methylome mapping of scarce biological material, including cell-free DNA and 10 pg input DNA.

How Flies See Motion.

How neurons detect the direction of motion is a prime example of neural computation: Motion vision is found in the visual systems of virtually all sighted animals, it is important for survival, and it requires interesting computations with well-defined linear and nonlinear processing steps-yet the whole process is of moderate complexity. The genetic methods available in the fruit fly Drosophila and the charting of a connectome of its visual system have led to rapid progress and unprecedented detail in our understanding of how neurons compute the direction of motion in this organism. The picture that emerged incorporates not only the identity, morphology, and synaptic connectivity of each neuron involved but also its neurotransmitters, its receptors, and their subcellular localization. Together with the neurons' membrane potential responses to visual stimulation, this information provides the basis for a biophysically realistic model of the circuit that computes the direction of visual motion.

Cancer screening with multicancer detection tests: A translational science review.

Multicancer detection (MCD) tests use a single, easily obtainable biospecimen, such as blood, to screen for more than one cancer concurrently. MCD tests can potentially be used to improve early cancer detection, including cancers that currently lack effective screening methods. However, these tests have unknown and unquantified benefits and harms. MCD tests differ from conventional cancer screening tests in that the organ responsible for a positive test is unknown, and a broad diagnostic workup may be necessary to confirm the location and type of underlying cancer. Among two prospective studies involving greater than 16,000 individuals, MCD tests identified those who had some cancers without currently recommended screening tests, including pancreas, ovary, liver, uterus, small intestine, oropharyngeal, bone, thyroid, and hematologic malignancies, at early stages. Reported MCD test sensitivities range from 27% to 95% but differ by organ and are lower for early stage cancers, for which treatment toxicity would be lowest and the potential for cure might be highest. False reassurance from a negative MCD result may reduce screening adherence, risking a loss in proven public health benefits from standard-of-care screening. Prospective clinical trials are needed to address uncertainties about MCD accuracy to detect different cancers in asymptomatic individuals, whether these tests can detect cancer sufficiently early for effective treatment and mortality reduction, the degree to which these tests may contribute to cancer overdiagnosis and overtreatment, whether MCD tests work equally well across all populations, and the appropriate diagnostic evaluation and follow-up for patients with a positive test.

Magic angle spinning NMR of proteins: high-frequency dynamic nuclear polarization and (1)H detection.

Magic angle spinning (MAS) NMR studies of amyloid and membrane proteins and large macromolecular complexes are an important new approach to structural biology. However, the applicability of these experiments, which are based on (13)C- and (15)N-detected spectra, would be enhanced if the sensitivity were improved. Here we discuss two advances that address this problem: high-frequency dynamic nuclear polarization (DNP) and (1)H-detected MAS techniques. DNP is a sensitivity enhancement technique that transfers the high polarization of exogenous unpaired electrons to nuclear spins via microwave irradiation of electron-nuclear transitions. DNP boosts NMR signal intensities by factors of 10(2) to 10(3), thereby overcoming NMR's inherent low sensitivity. Alternatively, it permits structural investigations at the nanomolar scale. In addition, (1)H detection is feasible primarily because of the development of MAS rotors that spin at frequencies of 40 to 60 kHz or higher and the preparation of extensively (2)H-labeled proteins.

Colorectal cancer statistics, 2023.

Colorectal cancer (CRC) is the second most common cause of cancer death in the United States. Every 3 years, the American Cancer Society provides an update of CRC statistics based on incidence from population-based cancer registries and mortality from the National Center for Health Statistics. In 2023, approximately 153,020 individuals will be diagnosed with CRC and 52,550 will die from the disease, including 19,550 cases and 3750 deaths in individuals younger than 50 years. The decline in CRC incidence slowed from 3%-4% annually during the 2000s to 1% annually during 2011-2019, driven partly by an increase in individuals younger than 55 years of 1%-2% annually since the mid-1990s. Consequently, the proportion of cases among those younger than 55 years increased from 11% in 1995 to 20% in 2019. Incidence since circa 2010 increased in those younger than 65 years for regional-stage disease by about 2%-3% annually and for distant-stage disease by 0.5%-3% annually, reversing the overall shift to earlier stage diagnosis that occurred during 1995 through 2005. For example, 60% of all new cases were advanced in 2019 versus 52% in the mid-2000s and 57% in 1995, before widespread screening. There is also a shift to left-sided tumors, with the proportion of rectal cancer increasing from 27% in 1995 to 31% in 2019. CRC mortality declined by 2% annually from 2011-2020 overall but increased by 0.5%-3% annually in individuals younger than 50 years and in Native Americans younger than 65 years. In summary, despite continued overall declines, CRC is rapidly shifting to diagnosis at a younger age, at a more advanced stage, and in the left colon/rectum. Progress against CRC could be accelerated by uncovering the etiology of rising incidence in generations born since 1950 and increasing access to high-quality screening and treatment among all populations, especially Native Americans.

Social determinants of health and US cancer screening interventions: A systematic review.

There remains a need to synthesize linkages between social determinants of health (SDOH) and cancer screening to reduce persistent inequities contributing to the US cancer burden. The authors conducted a systematic review of US-based breast, cervical, colorectal, and lung cancer screening intervention studies to summarize how SDOH have been considered in interventions and relationships between SDOH and screening. Five databases were searched for peer-reviewed research articles published in English between 2010 and 2021. The Covidence software platform was used to screen articles and extract data using a standardized template. Data items included study and intervention characteristics, SDOH intervention components and measures, and screening outcomes. The findings were summarized using descriptive statistics and narratives. The review included 144 studies among diverse population groups. SDOH interventions increased screening rates overall by a median of 8.4 percentage points (interquartile interval, 1.8-18.8 percentage points). The objective of most interventions was to increase community demand (90.3%) and access (84.0%) to screening. SDOH interventions related to health care access and quality were most prevalent (227 unique intervention components). Other SDOH, including educational, social/community, environmental, and economic factors, were less common (90, 52, 21, and zero intervention components, respectively). Studies that included analyses of health policy, access to care, and lower costs yielded the largest proportions of favorable associations with screening outcomes. SDOH were predominantly measured at the individual level. This review describes how SDOH have been considered in the design and evaluation of cancer screening interventions and effect sizes for SDOH interventions. Findings may guide future intervention and implementation research aiming to reduce US screening inequities.

Patient navigation across the cancer care continuum: An overview of systematic reviews and emerging literature.

Patient navigation is a strategy for overcoming barriers to reduce disparities and to improve access and outcomes. The aim of this umbrella review was to identify, critically appraise, synthesize, and present the best available evidence to inform policy and planning regarding patient navigation across the cancer continuum. Systematic reviews examining navigation in cancer care were identified in the Cochrane Central Register of Controlled Trials (CENTRAL), PubMed, Embase, Cumulative Index of Nursing and Allied Health (CINAHL), Epistemonikos, and Prospective Register of Systematic Reviews (PROSPERO) databases and in the gray literature from January 1, 2012, to April 19, 2022. Data were screened, extracted, and appraised independently by two authors. The JBI Critical Appraisal Checklist for Systematic Review and Research Syntheses was used for quality appraisal. Emerging literature up to May 25, 2022, was also explored to capture primary research published beyond the coverage of included systematic reviews. Of the 2062 unique records identified, 61 systematic reviews were included. Fifty-four reviews were quantitative or mixed-methods reviews, reporting on the effectiveness of cancer patient navigation, including 12 reviews reporting costs or cost-effectiveness outcomes. Seven qualitative reviews explored navigation needs, barriers, and experiences. In addition, 53 primary studies published since 2021 were included. Patient navigation is effective in improving participation in cancer screening and reducing the time from screening to diagnosis and from diagnosis to treatment initiation. Emerging evidence suggests that patient navigation improves quality of life and patient satisfaction with care in the survivorship phase and reduces hospital readmission in the active treatment and survivorship care phases. Palliative care data were extremely limited. Economic evaluations from the United States suggest the potential cost-effectiveness of navigation in screening programs.

Allosteric control of type I-A CRISPR-Cas3 complexes and establishment as effective nucleic acid detection and human genome editing tools.

Type I CRISPR-Cas systems typically rely on a two-step process to degrade DNA. First, an RNA-guided complex named Cascade identifies the complementary DNA target. The helicase-nuclease fusion enzyme Cas3 is then recruited in trans for processive DNA degradation. Contrary to this model, here, we show that type I-A Cascade and Cas3 function as an integral effector complex. We provide four cryoelectron microscopy (cryo-EM) snapshots of the Pyrococcus furiosus (Pfu) type I-A effector complex in different stages of DNA recognition and degradation. The HD nuclease of Cas3 is autoinhibited inside the effector complex. It is only allosterically activated upon full R-loop formation, when the entire targeted region has been validated by the RNA guide. The mechanistic insights inspired us to convert Pfu Cascade-Cas3 into a high-sensitivity, low-background, and temperature-activated nucleic acid detection tool. Moreover, Pfu CRISPR-Cas3 shows robust bi-directional deletion-editing activity in human cells, which could find usage in allele-specific inactivation of disease-causing mutations.

Highly Parallel Profiling of Cas9 Variant Specificity.

Determining the off-target cleavage profile of programmable nucleases is an important consideration for any genome editing experiment, and a number of Cas9 variants have been reported that improve specificity. We describe here tagmentation-based tag integration site sequencing (TTISS), an efficient, scalable method for analyzing double-strand breaks (DSBs) that we apply in parallel to eight Cas9 variants across 59 targets. Additionally, we generated thousands of other Cas9 variants and screened for variants with enhanced specificity and activity, identifying LZ3 Cas9, a high specificity variant with a unique +1 insertion profile. This comprehensive comparison reveals a general trade-off between Cas9 activity and specificity and provides information about the frequency of generation of +1 insertions, which has implications for correcting frameshift mutations.

Early Binaural Hearing: The Comparison of Temporal Differences at the Two Ears.

Many mammals, including humans, are exquisitely sensitive to tiny time differences between sounds at the two ears. These interaural time differences are an important source of information for sound detection, for sound localization in space, and for environmental awareness. Two brainstem circuits are involved in the initial temporal comparisons between the ears, centered on the medial and lateral superior olive. Cells in these nuclei, as well as their afferents, display a large number of striking physiological and anatomical specializations to enable submillisecond sensitivity. As such, they provide an important model system to study temporal processing in the central nervous system. We review the progress that has been made in characterizing these primary binaural circuits as well as the variety of mechanisms that have been proposed to underlie their function.

Multidisciplinary standards of care and recent progress in pancreatic ductal adenocarcinoma.

Despite tremendous gains in the molecular understanding of exocrine pancreatic cancer, the prognosis for this disease remains very poor, largely because of delayed disease detection and limited effectiveness of systemic therapies. Both incidence rates and mortality rates for pancreatic cancer have increased during the past decade, in contrast to most other solid tumor types. Recent improvements in multimodality care have substantially improved overall survival, local control, and metastasis-free survival for patients who have localized tumors that are amenable to surgical resection. The widening gap in prognosis between patients with resectable and unresectable or metastatic disease reinforces the importance of detecting pancreatic cancer sooner to improve outcomes. Furthermore, the developing use of therapies that target tumor-specific molecular vulnerabilities may offer improved disease control for patients with advanced disease. Finally, the substantial morbidity associated with pancreatic cancer, including wasting, fatigue, and pain, remains an under-addressed component of this disease, which powerfully affects quality of life and limits tolerance to aggressive therapies. In this article, the authors review the current multidisciplinary standards of care in pancreatic cancer with a focus on emerging concepts in pancreatic cancer detection, precision therapy, and survivorship.

Colorectal cancer statistics, 2020.

Colorectal cancer (CRC) is the second most common cause of cancer death in the United States. Every 3 years, the American Cancer Society provides an update of CRC occurrence based on incidence data (available through 2016) from population-based cancer registries and mortality data (through 2017) from the National Center for Health Statistics. In 2020, approximately 147,950 individuals will be diagnosed with CRC and 53,200 will die from the disease, including 17,930 cases and 3,640 deaths in individuals aged younger than 50 years. The incidence rate during 2012 through 2016 ranged from 30 (per 100,000 persons) in Asian/Pacific Islanders to 45.7 in blacks and 89 in Alaska Natives. Rapid declines in incidence among screening-aged individuals during the 2000s continued during 2011 through 2016 in those aged 65 years and older (by 3.3% annually) but reversed in those aged 50 to 64 years, among whom rates increased by 1% annually. Among individuals aged younger than 50 years, the incidence rate increased by approximately 2% annually for tumors in the proximal and distal colon, as well as the rectum, driven by trends in non-Hispanic whites. CRC death rates during 2008 through 2017 declined by 3% annually in individuals aged 65 years and older and by 0.6% annually in individuals aged 50 to 64 years while increasing by 1.3% annually in those aged younger than 50 years. Mortality declines among individuals aged 50 years and older were steepest among blacks, who also had the only decreasing trend among those aged younger than 50 years, and excluded American Indians/Alaska Natives, among whom rates remained stable. Progress against CRC can be accelerated by increasing access to guideline-recommended screening and high-quality treatment, particularly among Alaska Natives, and elucidating causes for rising incidence in young and middle-aged adults.

Programmable Inhibition and Detection of RNA Viruses Using Cas13.

The CRISPR effector Cas13 could be an effective antiviral for single-stranded RNA (ssRNA) viruses because it programmably cleaves RNAs complementary to its CRISPR RNA (crRNA). Here, we computationally identify thousands of potential Cas13 crRNA target sites in hundreds of ssRNA viral species that can potentially infect humans. We experimentally demonstrate Cas13's potent activity against three distinct ssRNA viruses: lymphocytic choriomeningitis virus (LCMV); influenza A virus (IAV); and vesicular stomatitis virus (VSV). Combining this antiviral activity with Cas13-based diagnostics, we develop Cas13-assisted restriction of viral expression and readout (CARVER), an end-to-end platform that uses Cas13 to detect and destroy viral RNA. We further screen hundreds of crRNAs along the LCMV genome to evaluate how conservation and target RNA nucleotide content influence Cas13's antiviral activity. Our results demonstrate that Cas13 can be harnessed to target a wide range of ssRNA viruses and CARVER's potential broad utility for rapid diagnostic and antiviral drug development.

Increased methane emissions from oil and gas following the Soviet Union's collapse.

Global atmospheric methane concentrations rose by 10 to 15 ppb/y in the 1980s before abruptly slowing to 2 to 8 ppb/y in the early 1990s. This period in the 1990s is known as the "methane slowdown" and has been attributed in part to the collapse of the former Soviet Union (USSR) in December 1991, which may have decreased the methane emissions from oil and gas operations. Here, we develop a methane plume detection system based on probabilistic deep learning and human-labeled training data. We use this method to detect methane plumes from Landsat 5 satellite observations over Turkmenistan from 1986 to 2011. We focus on Turkmenistan because economic data suggest it could account for half of the decline in oil and gas emissions from the former USSR. We find an increase in both the frequency of methane plume detections and the magnitude of methane emissions following the collapse of the USSR. We estimate a national loss rate from oil and gas infrastructure in Turkmenistan of more than 10% at times, which suggests the socioeconomic turmoil led to a lack of oversight and widespread infrastructure failure in the oil and gas sector. Our finding of increased oil and gas methane emissions from Turkmenistan following the USSR's collapse casts doubt on the long-standing hypothesis regarding the methane slowdown, begging the question: "what drove the 1992 methane slowdown?"

StratoLAMP: Label-free, multiplex digital loop-mediated isothermal amplification based on visual stratification of precipitate.

Multiplex, digital nucleic acid detections have important biomedical applications, but the multiplexity of existing methods is predominantly achieved using fluorescent dyes or probes, making the detection complicated and costly. Here, we present the StratoLAMP for label-free, multiplex digital loop-mediated isothermal amplification based on visual stratification of the precipitate byproduct. The StratoLAMP designates two sets of primers with different concentrations to achieve different precipitate yields when amplifying different nucleic acid targets. In the detection, deep learning image analysis is used to stratify the precipitate within each droplet and determine the encapsulated targets for nucleic acid quantification. We investigated the effect of the amplification reagents and process on the precipitate generation and optimized the assay conditions. We then implemented a deep-learning image analysis pipeline for droplet detection, achieving an overall accuracy of 94.3%. In the application, the StratoLAMP successfully achieved the simultaneous quantification of two nucleic acid targets with high accuracy. By eliminating the need for fluorescence, StratoLAMP represents a unique concept toward label-free, multiplex nucleic acid assays and an analytical tool with great cost-effectiveness.