Molecular cartography uncovers evolutionary and microenvironmental dynamics in sporadic colorectal tumors.

Colorectal cancer exhibits dynamic cellular and genetic heterogeneity during progression from precursor lesions toward malignancy. Analysis of spatial multi-omic data from 31 human colorectal specimens enabled phylogeographic mapping of tumor evolution that revealed individualized progression trajectories and accompanying microenvironmental and clonal alterations. Phylogeographic mapping ordered genetic events, classified tumors by their evolutionary dynamics, and placed clonal regions along global pseudotemporal progression trajectories encompassing the chromosomal instability (CIN+) and hypermutated (HM) pathways. Integrated single-cell and spatial transcriptomic data revealed recurring epithelial programs and infiltrating immune states along progression pseudotime. We discovered an immune exclusion signature (IEX), consisting of extracellular matrix regulators DDR1, TGFBI, PAK4, and DPEP1, that charts with CIN+ tumor progression, is associated with reduced cytotoxic cell infiltration, and shows prognostic value in independent cohorts. This spatial multi-omic atlas provides insights into colorectal tumor-microenvironment co-evolution, serving as a resource for stratification and targeted treatments.

Proteomic discovery of chemical probes that perturb protein complexes in human cells.

Most human proteins lack chemical probes, and several large-scale and generalizable small-molecule binding assays have been introduced to address this problem. How compounds discovered in such "binding-first" assays affect protein function, nonetheless, often remains unclear. Here, we describe a "function-first" proteomic strategy that uses size exclusion chromatography (SEC) to assess the global impact of electrophilic compounds on protein complexes in human cells. Integrating the SEC data with cysteine-directed activity-based protein profiling identifies changes in protein-protein interactions that are caused by site-specific liganding events, including the stereoselective engagement of cysteines in PSME1 and SF3B1 that disrupt the PA28 proteasome regulatory complex and stabilize a dynamic state of the spliceosome, respectively. Our findings thus show how multidimensional proteomic analysis of focused libraries of electrophilic compounds can expedite the discovery of chemical probes with site-specific functional effects on protein complexes in human cells.

Monoallelic expression of olfactory receptors.

The sense of smell collects vital information about the environment by detecting a multitude of chemical odorants. Breadth and sensitivity are provided by a huge number of chemosensory receptor proteins, including more than 1,400 olfactory receptors (ORs). Organizing the sensory information generated by these receptors so that it can be processed and evaluated by the central nervous system is a major challenge. This challenge is overcome by monogenic and monoallelic expression of OR genes. The single OR expressed by each olfactory sensory neuron determines the neuron's odor sensitivity and the axonal connections it will make to downstream neurons in the olfactory bulb. The expression of a single OR per neuron is accomplished by coupling a slow chromatin-mediated activation process to a fast negative-feedback signal that prevents activation of additional ORs. Singular OR activation is likely orchestrated by a network of interchromosomal enhancer interactions and large-scale changes in nuclear architecture.

Mimicking superinfection exclusion disrupts alphavirus infection and transmission in the yellow fever mosquito Aedes aegypti.

Multiple viruses, including pathogenic viruses, bacteriophages, and even plant viruses, cause a phenomenon termed superinfection exclusion whereby a currently infected cell is resistant to secondary infection by the same or a closely related virus. In alphaviruses, this process is thought to be mediated, at least in part, by the viral protease (nsP2) which is responsible for processing the nonstructural polyproteins (P123 and P1234) into individual proteins (nsP1-nsP4), forming the viral replication complex. Taking a synthetic biology approach, we mimicked this naturally occurring phenomenon by generating a superinfection exclusion-like state in Aedes aegypti mosquitoes, rendering them refractory to alphavirus infection. By artificially expressing Sindbis virus (SINV) and chikungunya virus (CHIKV) nsP2 in mosquito cells and transgenic mosquitoes, we demonstrated a reduction in both SINV and CHIKV viral replication rates in cells following viral infection as well as reduced infection prevalence, viral titers, and transmission potential in mosquitoes.

Darwinian evolution as a dynamical principle.

Darwinian evolution (DE)-biology's powerful process of adaptation-is remarkably different from other known dynamical processes. It is antithermodynamic, driving away from equilibrium; it has persisted for 3.5 billion years; and its target, fitness, can seem like "Just So" stories. For insights, we make a computational model. In the Darwinian Evolution Machine (DEM) model, resource-driven duplication and competition operate inside a cycle of search/compete/choose. We find the following: 1) DE requires multiorganism coexistence for its long-term persistence and ability to cross fitness valleys. 2) DE is driven by resource dynamics, like booms and busts, not just by mutational change. And, 3) fitness ratcheting requires a mechanistic separation between variation and selection steps, perhaps explaining biology's use of separate polymers, DNA and proteins.

Identification and characterization of transition metal-binding proteins and metabolites in the phloem sap of Brassica napus.

Transition metal (TM) distribution through the phloem is an essential part of plant metabolism and is required for systemic signaling and balancing source-to-sink relationships. Due to their reactivity, TMs are expected to occur in complexes within the phloem sap; however, metal speciation in the phloem sap remains largely unexplored. Here, we isolated phloem sap from Brassica napus and analyzed it via size exclusion chromatography (SEC) coupled online to sector-field ICP-MS. Our data identified known TM binding proteins and molecules including metallothioneins (MT), glutathione, and nicotianamine. While the main peak of all metals was low MW (∼1.5 kD), additional peaks ∼10-15 kD containing Cu, Fe, S and Zn were also found. Further physicochemical analyses of MTs with and without affinity tags corroborated that MTs can form complexes of diverse molecular weights. We also identified and characterized potential artifacts in the TM-biding ability of B. napus MTs between tagged and non-tagged MTs. That is, the native BnMT2 binds Zn, Cu and Fe, while MT3a and MT3b only bind Cu and Zn. In contrast, his-tagged MTs bind less Cu and were found to bind Co and Mn and aggregated to oligomeric forms to a greater extent compared to the phloem sap. Our data indicates that TM chemistry in the phloem sap is more complex than previously anticipated and that more systematic analyses are needed to establish the precise speciation of TM and TM-ligand complexes within the phloem sap.

Metabolic adaptations leading to an enhanced lignification in wheat roots under salinity stress.

Analysis of salinity tolerance processes in wheat has focused on salt exclusion from shoots while root phenotypes have received limited attention. Here, we consider the varying phenotypic response of four bread wheat varieties that differ in their type and degree of salt tolerance and assess their molecular responses to salinity and changes in root cell wall lignification. These varieties were Westonia introgressed with Nax1 and Nax2 root sodium transporters (HKT1;4-A and HKT1;5-A) that reduce Na+ accumulation in leaves, as well as the 'tissue tolerant' Portuguese landrace Mocho de Espiga Branca that has a mutation in the homologous gene HKT1;5-D and has high Na+ concentration in leaves. These three varieties were compared with the relatively more salt-sensitive cultivar Gladius. Through the use of root histochemical analysis, ion concentrations, as well as differential proteomics and targeted metabolomics, we provide an integrated view of the wheat root response to salinity. We show different metabolic re-arrangements in energy conversion, primary metabolic machinery and phenylpropanoid pathway leading to monolignol production in a genotype and genotype by treatment-dependent manner that alters the extent and localisation of root lignification which correlated with an improved capacity of wheat roots to cope better under salinity stress.

Identification of complier and noncomplier average causal effects in the presence of latent missing-at-random (LMAR) outcomes: a unifying view and choices of assumptions.

The study of treatment effects is often complicated by noncompliance and missing data. In the one-sided noncompliance setting where of interest are the complier and noncomplier average causal effects, we address outcome missingness of the latent missing at random type (LMAR, also known as latent ignorability). That is, conditional on covariates and treatment assigned, the missingness may depend on compliance type. Within the instrumental variable (IV) approach to noncompliance, methods have been proposed for handling LMAR outcome that additionally invoke an exclusion restriction-type assumption on missingness, but no solution has been proposed for when a non-IV approach is used. This article focuses on effect identification in the presence of LMAR outcomes, with a view to flexibly accommodate different principal identification approaches. We show that under treatment assignment ignorability and LMAR only, effect nonidentifiability boils down to a set of two connected mixture equations involving unidentified stratum-specific response probabilities and outcome means. This clarifies that (except for a special case) effect identification generally requires two additional assumptions: a specific missingness mechanism assumption and a principal identification assumption. This provides a template for identifying effects based on separate choices of these assumptions. We consider a range of specific missingness assumptions, including those that have appeared in the literature and some new ones. Incidentally, we find an issue in the existing assumptions, and propose a modification of the assumptions to avoid the issue. Results under different assumptions are illustrated using data from the Baltimore Experience Corps Trial.

Japanese encephalitis virus inhibits superinfection of Zika virus in cells by the NS2B protein.

Superinfection exclusion (SIE) is a phenomenon in which a preexisting infection prevents a secondary infection. SIE has been described for several flaviviruses, such as West Nile virus vs Nhumirim virus and Dengue virus vs yellow fever virus. Zika virus (ZIKV) is an emerging flavivirus posing threats to human health. The SIE between ZIKV and Japanese encephalitis virus (JEV) is investigated in this study. Our results demonstrate for the first time that JEV inhibits ZIKV infection in both mammalian and mosquito cells, whether co-infects or subsequently infects after ZIKV. The exclusion effect happens at the stage of ZIKV RNA replication. Further studies show that the expression of JEV NS2B protein is sufficient to inhibit the replication of ZIKV, and the outer membrane region of NS2B (46-103 aa) is responsible for this SIE. JEV infection and NS2B expression also inhibit the infection of the vesicular stomatitis virus. In summary, our study characterized a SIE caused by JEV NS2B. This may have potential applications in the prevention and treatment of ZIKV or other RNA viruses.IMPORTANCEThe reemerged Zika virus (ZIKV) has caused severe symptoms in humans and poses a continuous threat to public health. New vaccines or antiviral agents need to be developed to cope with possible future pandemics. In this study, we found that infection of Japanese encephalitis virus (JEV) or expression of NS2B protein well inhibited the replication of ZIKV. It is worth noting that both the P3 strain and vaccine strain SA14-14-2 of JEV exhibited significant inhibitory effects on ZIKV. Additionally, the JEV NS2B protein also had an inhibitory effect on vesicular stomatitis virus infection, suggesting that it may be a broad-spectrum antiviral factor. These findings provide a new way of thinking about the prevention and treatment of ZIKV.

In vitro higher-order oligomeric assembly of the respiratory syncytial virus M2-1 protein with longer RNAs.

The respiratory syncytial virus (RSV) M2-1 protein is a transcriptional antitermination factor crucial for efficiently synthesizing multiple full-length viral mRNAs. During RSV infection, M2-1 exists in a complex with mRNA within cytoplasmic compartments called inclusion body-associated granules (IBAGs). Prior studies showed that M2-1 can bind along the entire length of viral mRNAs instead of just gene-end (GE) sequences, suggesting that M2-1 has more sophisticated RNA recognition and binding characteristics. Here, we analyzed the higher oligomeric complexes formed by M2-1 and RNAs in vitro using size exclusion chromatography (SEC), electrophoretic mobility shift assays (EMSA), negative stain electron microscopy (EM), and mutagenesis. We observed that the minimal RNA length for such higher oligomeric assembly is about 14 nucleotides for polyadenine sequences, and longer RNAs exhibit distinct RNA-induced binding modality to M2-1, leading to enhanced particle formation frequency and particle homogeneity as the local RNA concentration increases. We showed that particular cysteine residues of the M2-1 cysteine-cysteine-cystine-histidine (CCCH) zinc-binding motif are essential for higher oligomeric assembly. Furthermore, complexes assembled with long polyadenine sequences remain unaffected when co-incubated with ribonucleases or a zinc chelation agent. Our study provided new insights into the higher oligomeric assembly of M2-1 with longer RNA.IMPORTANCERespiratory syncytial virus (RSV) causes significant respiratory infections in infants, the elderly, and immunocompromised individuals. The virus forms specialized compartments to produce genetic material, with the M2-1 protein playing a pivotal role. M2-1 acts as an anti-terminator in viral transcription, ensuring the creation of complete viral mRNA and associating with both viral and cellular mRNA. Our research focuses on understanding M2-1's function in viral mRNA synthesis by modeling interactions in a controlled environment. This approach is crucial due to the challenges of studying these compartments in vivo. Reconstructing the system in vitro uncovers structural and biochemical aspects and reveals the potential functions of M2-1 and its homologs in related viruses. Our work may contribute to identifying targets for antiviral inhibitors and advancing RSV infection treatment.

Safety concern of recombination between self-amplifying mRNA vaccines and viruses is mitigated in vivo.

Self-amplifying mRNA (SAM) vaccines can be rapidly deployed in the event of disease outbreaks. A legitimate safety concern is the potential for recombination between alphavirus-based SAM vaccines and circulating viruses. This theoretical risk needs to be assessed in the regulatory process for SAM vaccine approval. Herein, we undertake extensive in vitro and in vivo assessments to explore recombination between SAM vaccine and a wide selection of alphaviruses and a coronavirus. SAM vaccines were found to effectively limit alphavirus co-infection through superinfection exclusion, although some co-replication was still possible. Using sensitive cell-based assays, replication-competent alphavirus chimeras were generated in vitro as a result of rare, but reproducible, RNA recombination events. The chimeras displayed no increased fitness in cell culture. Viable alphavirus chimeras were not detected in vivo in C57BL/6J, Rag1-/- and Ifnar-/- mice, in which high levels of SAM vaccine and alphavirus co-replicated in the same tissue. Furthermore, recombination between a SAM-spike vaccine and a swine coronavirus was not observed. In conclusion we state that although the ability of SAM vaccines to recombine with alphaviruses might be viewed as an environmental safety concern, several key factors substantially mitigate against in vivo emergence of chimeric viruses from SAM vaccine recipients.

Size-exclusion chromatography combined with DIA-MS enables deep proteome profiling of extracellular vesicles from melanoma plasma and serum.

Extracellular vesicles (EVs) are important players in melanoma progression, but their use as clinical biomarkers has been limited by the difficulty of profiling blood-derived EV proteins with high depth of coverage, the requirement for large input amounts, and complex protocols. Here, we provide a streamlined and reproducible experimental workflow to identify plasma- and serum- derived EV proteins of healthy donors and melanoma patients using minimal amounts of sample input. SEC-DIA-MS couples size-exclusion chromatography to EV concentration and deep-proteomic profiling using data-independent acquisition. From as little as 200 µL of plasma per patient in a cohort of three healthy donors and six melanoma patients, we identified and quantified 2896 EV-associated proteins, achieving a 3.5-fold increase in depth compared to previously published melanoma studies. To compare the EV-proteome to unenriched blood, we employed an automated workflow to deplete the 14 most abundant proteins from plasma and serum and thereby approximately doubled protein group identifications versus native blood. The EV proteome diverged from corresponding unenriched plasma and serum, and unlike the latter, separated healthy donor and melanoma patient samples. Furthermore, known melanoma markers, such as MCAM, TNC, and TGFBI, were upregulated in melanoma EVs but not in depleted melanoma plasma, highlighting the specific information contained in EVs. Overall, EVs were significantly enriched in intact membrane proteins and proteins related to SNARE protein interactions and T-cell biology. Taken together, we demonstrated the increased sensitivity of an EV-based proteomic workflow that can be easily applied to larger melanoma cohorts and other indications.

Structural basis for host recognition and superinfection exclusion by bacteriophage T5.

A key but poorly understood stage of the bacteriophage life cycle is the binding of phage receptor-binding proteins (RBPs) to receptors on the host cell surface, leading to injection of the phage genome and, for lytic phages, host cell lysis. To prevent secondary infection by the same or a closely related phage and nonproductive phage adsorption to lysed cell fragments, superinfection exclusion (SE) proteins can prevent the binding of RBPs via modulation of the host receptor structure in ways that are also unclear. Here, we present the cryogenic electron microscopy (cryo-EM) structure of the phage T5 outer membrane (OM) receptor FhuA in complex with the T5 RBP pb5, and the crystal structure of FhuA complexed to the OM SE lipoprotein Llp. Pb5 inserts four loops deeply into the extracellular lumen of FhuA and contacts the plug but does not cause any conformational changes in the receptor, supporting the view that DNA translocation does not occur through the lumen of OM channels. The FhuA-Llp structure reveals that Llp is periplasmic and binds to a nonnative conformation of the plug of FhuA, causing the inward folding of two extracellular loops via "reverse" allostery. The inward-folded loops of FhuA overlap with the pb5 binding site, explaining how Llp binding to FhuA abolishes further infection of Escherichia coli by phage T5 and suggesting a mechanism for SE via the jamming of TonB-dependent transporters by small phage lipoproteins.

A fast and sensitive size-exclusion chromatography method for plasma extracellular vesicle proteomic analysis.

Extracellular vesicles (EVs) carry diverse biomolecules derived from their parental cells, making their components excellent biomarker candidates. However, purifying EVs is a major hurdle in biomarker discovery since current methods require large amounts of samples, are time-consuming and typically have poor reproducibility. Here we describe a simple, fast, and sensitive EV fractionation method using size exclusion chromatography (SEC) on a fast protein liquid chromatography (FPLC) system. Our method uses a Superose 6 Increase 5/150, which has a bed volume of 2.9 mL. The FPLC system and small column size enable reproducible separation of only 50 µL of human plasma in 15 min. To demonstrate the utility of our method, we used longitudinal samples from a group of individuals who underwent intense exercise. A total of 838 proteins were identified, of which, 261 were previously characterized as EV proteins, including classical markers, such as cluster of differentiation (CD)9 and CD81. Quantitative analysis showed low technical variability with correlation coefficients greater than 0.9 between replicates. The analysis captured differences in relevant EV proteins involved in response to physical activity. Our method enables fast and sensitive fractionation of plasma EVs with low variability, which will facilitate biomarker studies in large clinical cohorts.

Filling the gaps in peptide maps with a platform assay for top-down characterization of purified protein samples.

Liquid chromatography-mass spectrometry (LC-MS) intact mass analysis and LC-MS/MS peptide mapping are decisional assays for developing biological drugs and other commercial protein products. Certain PTM types, such as truncation and oxidation, increase the difficulty of precise proteoform characterization owing to inherent limitations in peptide and intact protein analyses. Top-down MS (TDMS) can resolve this ambiguity via fragmentation of specific proteoforms. We leveraged the strengths of flow-programmed (fp) denaturing online buffer exchange (dOBE) chromatography, including robust automation, relatively high ESI sensitivity, and long MS/MS window time, to support a TDMS platform for industrial protein characterization. We tested data-dependent (DDA) and targeted strategies using 14 different MS/MS scan types featuring combinations of collisional- and electron-based fragmentation as well as proton transfer charge reduction. This large, focused dataset was processed using a new software platform, named TDAcquireX, that improves proteoform characterization through TDMS data aggregation. A DDA-based workflow provided objective identification of αLac truncation proteoforms with a two-termini clipping search. A targeted TDMS workflow facilitated the characterization of αLac oxidation positional isomers. This strategy relied on using sliding window-based fragment ion deconvolution to generate composite proteoform spectral match (cPrSM) results amenable to fragment noise filtering, which is a fundamental enhancement relevant to TDMS applications generally.

Long-term rain exclusion in a Mediterranean forest: response of physiological and physico-chemical traits of Quercus pubescens across seasons.

With climate change, an aggravation in summer drought is expected in the Mediterranean region. To assess the impact of such a future scenario, we compared the response of Quercus pubescens, a drought-resistant deciduous oak species, to long-term amplified drought (AD) (partial rain exclusion in natura for 10 years) and natural drought (ND). We studied leaf physiological and physico-chemical trait responses to ND and AD over the seasonal cycle, with a focus on chemical traits including major groups of central (photosynthetic pigments and plastoquinones) and specialized (tocochromanols, phenolic compounds, and cuticular waxes) metabolites. Seasonality was the main driver of all leaf traits, including cuticular triterpenoids, which were highly concentrated in summer, suggesting their importance to cope with drought and thermal stress periods. Under AD, trees not only reduced CO2 assimilation (-42%) in summer and leaf concentrations of some phenolic compounds and photosynthetic pigments (carotenoids from the xanthophyll cycle) but also enhanced the levels of other photosynthetic pigments (chlorophylls, lutein, and neoxanthin) and plastochromanol-8, an antioxidant located in chloroplasts. Overall, the metabolomic adjustments across seasons and drought conditions reinforce the idea that Q. pubescens is highly resistant to drought although significant losses of antioxidant defenses and photoprotection were identified under AD.

Size exclusion experiment in a grassland field unravels top-down control of the soil fauna on microbial community assembly.

Highly diverse and abundant organisms coexist in soils. However, the contribution of biotic interactions between soil organisms to microbial community assembly remains to be explored. Here, we assess the extent to which soil fauna can shape microbial community assembly using an exclusion experiment in a grassland field to sort soil biota based on body size. After 1 year, the exclusion of larger fauna favoured phagotrophic protists, with increases up to 32% in their proportion compared to the no-mesh treatment. In contrast, members of the bacterial community and to a lesser extent of the fungal community were negatively impacted. Shifts in bacterial but not in fungal communities were best explained by the response of the protistan community to exclusion. Our findings provide empirical evidence of top-down control on the soil microbial communities and underline the importance of integrating higher trophic levels for a better understanding of the soil microbiome assembly.

Grazing herbivores reduce herbaceous biomass and fire activity across African savannas.

Fire and herbivory interact to alter ecosystems and carbon cycling. In savannas, herbivores can reduce fire activity by removing grass biomass, but the size of these effects and what regulates them remain uncertain. To examine grazing effects on fuels and fire regimes across African savannas, we combined data from herbivore exclosure experiments with remotely sensed data on fire activity and herbivore density. We show that, broadly across African savannas, grazing herbivores substantially reduce both herbaceous biomass and fire activity. The size of these effects was strongly associated with grazing herbivore densities, and surprisingly, was mostly consistent across different environments. A one-zebra increase in herbivore biomass density (~100 kg/km2 of metabolic biomass) resulted in a ~53 kg/ha reduction in standing herbaceous biomass and a ~0.43 percentage point reduction in burned area. Our results indicate that fire models can be improved by incorporating grazing effects on grass biomass.

Phenotypic and spatial heterogeneity of CD8+ tumour infiltrating lymphocytes.

CD8+ T cells are the workhorses executing adaptive anti-tumour response, and targets of various cancer immunotherapies. Latest advances have unearthed the sheer heterogeneity of CD8+ tumour infiltrating lymphocytes, and made it increasingly clear that the bulk of the endogenous and therapeutically induced tumour-suppressive momentum hinges on a particular selection of CD8+ T cells with advantageous attributes, namely the memory and stem-like exhausted subsets. A scrutiny of the contemporary perception of CD8+ T cells in cancer and the subgroups of interest along with the factors arbitrating their infiltration contextures, presented herein, may serve as the groundwork for future endeavours to probe further into the regulatory networks underlying their differentiation and migration, and optimise T cell-based immunotherapies accordingly.

Medicaid Inmate Exclusion Policy and Infectious Diseases Care for Justice-Involved Populations.

The Medicaid Inmate Exclusion Policy (MIEP) prohibits using federal funds for ambulatory care services and medications (including for infectious diseases) for incarcerated persons. More than one quarter of states, including California and Massachusetts, have asked the federal government for authority to waive the MIEP. To improve health outcomes and continuation of care, those states seek to cover transitional care services provided to persons in the period before release from incarceration. The Massachusetts Sheriffs' Association, Massachusetts Department of Correction, Executive Office of Health and Human Services, and University of Massachusetts Chan Medical School have collaborated to improve infectious disease healthcare service provision before and after release from incarceration. They seek to provide stakeholders working at the intersection of criminal justice and healthcare with tools to advance Medicaid policy and improve treatment and prevention of infectious diseases for persons in jails and prisons by removing MIEP barriers through Section 1115 waivers.