Proteomics-The State of the Field: The Definition and Analysis of Proteomes Should Be Based in Reality, Not Convenience.

With growing recognition and acknowledgement of the genuine complexity of proteomes, we are finally entering the post-proteogenomic era. Routine assessment of proteomes as inferred correlates of gene sequences (i.e., canonical 'proteins') cannot provide the necessary critical analysis of systems-level biology that is needed to understand underlying molecular mechanisms and pathways or identify the most selective biomarkers and therapeutic targets. These critical requirements demand the analysis of proteomes at the level of proteoforms/protein species, the actual active molecular players. Currently, only highly refined integrated or integrative top-down proteomics (iTDP) enables the analytical depth necessary to provide routine, comprehensive, and quantitative proteome assessments across the widest range of proteoforms inherent to native systems. Here we provide a broad perspective of the field, taking in historical and current realities, to establish a more balanced understanding of where the field has come from (in particular during the ten years since Proteomes was launched), current issues, and how things likely need to proceed if necessary deep proteome analyses are to succeed. We base this in our firm belief that the best proteomic analyses reflect, as closely as possible, the native sample at the moment of sampling. We also seek to emphasise that this and future analytical approaches are likely best based on the broad recognition and exploitation of the complementarity of currently successful approaches. This also emphasises the need to continuously evaluate and further optimize established approaches, to avoid complacency in thinking and expectations but also to promote the critical and careful development and introduction of new approaches, most notably those that address proteoforms. Above all, we wish to emphasise that a rigorous focus on analytical quality must override current thinking that largely values analytical speed; the latter would certainly be nice, if only proteoforms could thus be effectively, routinely, and quantitatively assessed. Alas, proteomes are composed of proteoforms, not molecular species that can be amplified or that directly mirror genes (i.e., 'canonical'). The problem is hard, and we must accept and address it as such, but the payoff in playing this longer game of rigorous deep proteome analyses is the promise of far more selective biomarkers, drug targets, and truly personalised or even individualised medicine.

[Structures, aims and needs of early career scientists at the German Center for Mental Health]. / Strukturen, Ziele und Bedürfnisse der Early Career Scientists am Deutschen Zentrum für Psychische Gesundheit.

BACKGROUND: Early career scientists (ECS) are agents of change and driving forces in the promotion of mental health. The German Center for Mental Health (DZPG) is a powerful initiative to guide and support careers in the field of mental health. OBJECTIVE: The DZPG aims to make investments to educate, engage, excite, and empower ECS in an interdisciplinary and interinstitutional scientific community. STRUCTURES, TOPICS AND INITIATIVES: To achieve this, the ECS Board at the DZPG plays a central role and consists of 18 elected ECS representatives. The ECS culture gives members the right of voice and embraces bottom-to-top ideas and acknowledges autonomy and co-determination. The DZPG academy was developed to facilitate communication and networking and encourage collaboration among ECS members. The DZPG also navigates several key issues, such as equality, diversity, inclusion, family friendliness and work-life balance, which are essential for a functioning research landscape. The DZPG also extends opportunities to ECS to develop skills and competencies that are essential for contemporary ECS. It complements nationwide support for ECS with funding opportunities, mental health support at work, careers advice and guidance activities. Importantly, the ECS Board is committed to patient and public involvement and engagement, scientific communication and knowledge transfer to multiple settings. CONCLUSION: The DZPG will contribute to fostering ECS training programs for student and academic exchanges, collaborative research, and pooling of resources to acquire grants and scholarships. It will also support the establishment of hubs for ECS networks and promote the expansion of international competence of ECS in Germany.

Exploring the Role of Complexity in Health Care Technology Bottom-Up Innovations: Multiple-Case Study Using the Nonadoption, Abandonment, Scale-Up, Spread, and Sustainability Complexity Assessment Tool.

BACKGROUND: New digital technology presents new challenges to health care on multiple levels. There are calls for further research that considers the complex factors related to digital innovations in complex health care settings to bridge the gap when moving from linear, logistic research to embracing and testing the concept of complexity. The nonadoption, abandonment, scale-up, spread, and sustainability (NASSS) framework was developed to help study complexity in digital innovations. OBJECTIVE: This study aims to investigate the role of complexity in the development and deployment of innovations by retrospectively assessing challenges to 4 digital health care innovations initiated from the bottom up. METHODS: A multicase retrospective, deductive, and explorative analysis using the NASSS complexity assessment tool LONG was conducted. In total, 4 bottom-up innovations developed in Region Västra Götaland in Sweden were explored and compared to identify unique and shared complexity-related challenges. RESULTS: The analysis resulted in joint insights and individual learning. Overall, the complexity was mostly found outside the actual innovation; more specifically, it related to the organization's readiness to integrate new innovations, how to manage and maintain innovations, and how to finance them. The NASSS framework sheds light on various perspectives that can either facilitate or hinder the adoption, scale-up, and spread of technological innovations. In the domain of condition or diagnosis, a well-informed understanding of the complexity related to the condition or illness (diabetes, cancer, bipolar disorders, and schizophrenia disorders) is of great importance for the innovation. The value proposition needs to be clearly described early to enable an understanding of costs and outcomes. The questions in the NASSS complexity assessment tool LONG were sometimes difficult to comprehend, not only from a language perspective but also due to a lack of understanding of the surrounding organization's system and its setting. CONCLUSIONS: Even when bottom-up innovations arise within the same support organization, the complexity can vary based on the developmental phase and the unique characteristics of each project. Identifying, defining, and understanding complexity may not solve the issues but substantially improves the prospects for successful deployment. Successful innovation within complex organizations necessitates an adaptive leadership and structures to surmount cultural resistance and organizational impediments. A rigid, linear, and stepwise approach risks disregarding interconnected variables and dependencies, leading to suboptimal outcomes. Success lies in embracing the complexity with its uncertainty, nurturing creativity, and adopting a nonlinear methodology that accommodates the iterative nature of innovation processes within complex organizations.

Effectiveness of population-based recovery actions for threatened southern mountain caribou.

Habitat loss is affecting many species, including the southern mountain caribou (Rangifer tarandus caribou) population in western North America. Over the last half century, this threatened caribou population's range and abundance have dramatically contracted. An integrated population model was used to analyze 51 years (1973-2023) of demographic data from 40 southern mountain caribou subpopulations to assess the effectiveness of population-based recovery actions at increasing population growth. Reducing potential limiting factors on threatened caribou populations offered a rare opportunity to identify the causes of decline and assess methods of recovery. Southern mountain caribou abundance declined by 51% between 1991 and 2023, and 37% of subpopulations were functionally extirpated. Wolf reduction was the only recovery action that consistently increased population growth when applied in isolation, and combinations of wolf reductions with maternal penning or supplemental feeding provided rapid growth but were applied to only four subpopulations. As of 2023, recovery actions have increased the abundance of southern mountain caribou by 52%, compared to a simulation with no interventions. When predation pressure was reduced, rapid population growth was observed, even under contemporary climate change and high levels of habitat loss. Unless predation is reduced, caribou subpopulations will continue to be extirpated well before habitat conservation and restoration can become effective.

Blood donation projections using hierarchical time series forecasting: the case of Zimbabwe's national blood bank.

BACKGROUND: The discrepancy between blood supply and demand requires accurate forecasts of the blood supply at any blood bank. Accurate blood donation forecasting gives blood managers empirical evidence in blood inventory management. The study aims to model and predict blood donations in Zimbabwe using hierarchical time series. The modelling technique allows one to identify, say, a declining donor category, and in that way, the method offers feasible and targeted solutions for blood managers to work on. METHODS: The monthly blood donation data covering the period 2007 to 2018, collected from the National Blood Service Zimbabwe (NBSZ) was used. The data was disaggregated by gender and blood groups types within each gender category. The model validation involved utilising actual blood donation data from 2019 and 2020. The model's performance was evaluated through the Mean Absolute Percentage Error (MAPE), uncovering expected and notable discrepancies during the Covid-19 pandemic period only. RESULTS: Blood group O had the highest monthly yield mean of 1507.85 and 1230.03 blood units for male and female donors, respectively. The top-down forecasting proportions (TDFP) under ARIMA, with a MAPE value of 11.30, was selected as the best approach and the model was then used to forecast future blood donations. The blood donation predictions for 2019 had a MAPE value of 14.80, suggesting alignment with previous years' donations. However, starting in April 2020, the Covid-19 pandemic disrupted blood collection, leading to a significant decrease in blood donation and hence a decrease in model accuracy. CONCLUSIONS: The gradual decrease in future blood donations exhibited by the predictions calls for blood authorities in Zimbabwe to develop interventions that encourage blood donor retention and regular donations. The impact of the Covid-19 pandemic distorted the blood donation patterns such that the developed model did not capture the significant drop in blood donations during the pandemic period. Other shocks such as, a surge in global pandemics and other disasters, will inevitably affect the blood donation system. Thus, forecasting future blood collections with a high degree of accuracy requires robust mathematical models which factor in, the impact of various shocks to the system, on short notice.

Plant growth-defense trade-offs are general across interactions with fungal, insect, and mammalian consumers.

Plants face trade-offs between allocating resources to growth, while also defending against herbivores or pathogens. Species differences along defense trade-off axes may promote coexistence and maintain diversity. However, few studies of plant communities have simultaneously compared defense trade-offs against an array of herbivores and pathogens for which defense investment may differ, and even fewer have been conducted in the complex natural communities in which these interactions unfold. We tested predictions about the role of defense trade-offs with competition and growth in diversity maintenance by tracking plant species abundance in a field experiment that removed individual consumer groups (mammals, arthropods, fungi) and added nutrients. Consistent with a growth-defense trade-off, plant species that increased in mass in response to nutrient addition also increased when consumers were removed. This growth-defense trade-off occurred for all consumer groups studied. Nutrient addition reduced plant species richness, which is consistent with trade-off theory. Removing foliar fungi increased plant diversity via increased species evenness, whereas removal of other consumer groups had little effect on diversity, counter to expectations. Thus, while growth-defense trade-offs are general across consumer groups, this trade-off observed in wild plant communities does not necessarily support plant diversity maintenance.

Between bodily action and conventionalized structure: The neural mechanisms of constructed action in sign language comprehension.

Sign languages (SLs) are expressed through different bodily actions, ranging from re-enactment of physical events (constructed action, CA) to sequences of lexical signs with internal structure (plain telling, PT). Despite the prevalence of CA in signed interactions and its significance for SL comprehension, its neural dynamics remain unexplored. We examined the processing of different types of CA (subtle, reduced, and overt) and PT in 35 adult deaf or hearing native signers. The electroencephalographic-based processing of signed sentences with incongruent targets was recorded. Attenuated N300 and early N400 were observed for CA in deaf but not in hearing signers. No differences were found between sentences with CA types in all signers, suggesting a continuum from PT to overt CA. Deaf signers focused more on body movements; hearing signers on faces. We conclude that CA is processed less effortlessly than PT, arguably because of its strong focus on bodily actions.

Seabird guano inputs increase impacts from introduced mammals on the native plants and animals of an oceanic island.

Seabirds create fluxes of nutrients from marine to terrestrial ecosystems that influence the food webs of small islands. We investigated how guano inputs shape terrestrial food webs by comparing species of selected plant and animal species in a red-footed booby colony in Mona Island (Puerto Rico, Caribbean Sea), to sites of the island lacking guano inputs. We quantified guano deposition and its relationship to plant biomass production, fecundity and density, as well as the activity of native and introduced animal species. In general, guano inputs increased the gross primary plant productivity, size, and fecundity by twofold. Guano inputs were also associated with twofold increases in density of Anole lizards, but also to increases in the activity of introduced pigs (> 500%), goats (> 30%), and cats (> 500%), which negatively impact native species. In particular, elevated pig and cat activity within the booby colony was correlated with lower activity of endemic ground lizards and of introduced rats. Our results also suggest that severe droughts associated with climate change exacerbate the negative effects that introduced species have on vegetation and reduce the positive effects of seabird guano inputs. Our findings underscore the importance of allochthonous guano inputs in subsidizing plant productivity and native and endemic species in small oceanic islands, but also in increasing the negative impacts of introduced mammals. Management and conservation efforts should focus on the exclusion (or eradication) of introduced mammals, particularly pigs and goats, from remnant seabird colonies in Mona Island.

Bioprinting of human dermal microtissues precursors as building blocks for endogenousin vitroconnective tissue manufacturing.

The advent of 3D bioprinting technologies in tissue engineering has unlocked the potential to fabricatein vitrotissue models, overcoming the constraints associated with the shape limitations of preformed scaffolds. However, achieving an accurate mimicry of complex tissue microenvironments, encompassing cellular and biochemical components, and orchestrating their supramolecular assembly to form hierarchical structures while maintaining control over tissue formation, is crucial for gaining deeper insights into tissue repair and regeneration. Building upon our expertise in developing competent three-dimensional tissue equivalents (e.g. skin, gut, cervix), we established a two-step bottom-up approach involving the dynamic assembly of microtissue precursors (µTPs) to generate macroscopic functional tissue composed of cell-secreted extracellular matrix (ECM). To enhance precision and scalability, we integrated extrusion-based bioprinting technology into our established paradigm to automate, control and guide the coherent assembly ofµTPs into predefined shapes. Compared to cell-aggregated bioink, ourµTPs represent a functional unit where cells are embedded in their specific ECM.µTPs were derived from human dermal fibroblasts dynamically seeded onto gelatin-based microbeads. After 9 days,µTPs were suspended (50% v/v) in Pluronic-F127 (30% w/v) (µTP:P30), and the obtained formulation was loaded as bioink into the syringe of the Dr.INVIVO-4D6 extrusion based bioprinter.µTP:P30 bioink showed shear-thinning behavior and temperature-dependent viscosity (gel atT> 30 °C), ensuringµTPs homogenous dispersion within the gel and optimal printability. The bioprinting involved extruding several geometries (line, circle, and square) into Pluronic-F127 (40% w/v) (P40) support bath, leveraging its shear-recovery property. P40 effectively held the bioink throughout and after the bioprinting procedure, untilµTPs fused into a continuous connective tissue.µTPs fusion dynamics was studied over 8 days of culture, while the resulting endogenous construct underwent 28 days culture. Histological, immunofluorescence analysis, and second harmonic generation reconstruction revealed an increase in endogenous collagen and fibronectin production within the bioprinted construct, closely resembling the composition of the native connective tissues.

Utilizing bioprinting to engineer spatially organized tissues from the bottom-up.

In response to the growing demand for organ substitutes, tissue engineering has evolved significantly. However, it is still challenging to create functional tissues and organs. Tissue engineering from the 'bottom-up' is promising on solving this problem due to its ability to construct tissues with physiological complexity. The workflow of this strategy involves two key steps: the creation of building blocks, and the subsequent assembly. There are many techniques developed for the two pivotal steps. Notably, bioprinting is versatile among these techniques and has been widely used in research. With its high level of automation, bioprinting has great capacity in engineering tissues with precision and holds promise to construct multi-material tissues. In this review, we summarize the techniques applied in fabrication and assembly of building blocks. We elaborate mechanisms and applications of bioprinting, particularly in the 'bottom-up' strategy. We state our perspectives on future trends of bottom-up tissue engineering, hoping to provide useful reference for researchers in this field.

Coherent long-term body-size responses across all Northwest Atlantic herring populations to warming and environmental change despite contrasting harvest and ecological factors.

Body size is a key component of individual fitness and an important factor in the structure and functioning of populations and ecosystems. Disentangling the effects of environmental change, harvest and intra- and inter-specific trophic effects on body size remains challenging for populations in the wild. Herring in the Northwest Atlantic provide a strong basis for evaluating hypotheses related to these drivers given that they have experienced significant warming and harvest over the past century, while also having been exposed to a wide range of other selective constraints across their range. Using data on mean length-at-age 4 for the sixteen principal populations over a period of 53 cohorts (1962-2014), we fitted a series of empirical models for temporal and between-population variation in the response to changes in sea surface temperature. We find evidence for a unified cross-population response in the form of a parabolic function according to which populations in naturally warmer environments have responded more negatively to increasing temperature compared with those in colder locations. Temporal variation in residuals from this function was highly coherent among populations, further suggesting a common response to a large-scale environmental driver. The synchrony observed in this study system, despite strong differences in harvest and ecological histories among populations and over time, clearly indicates a dominant role of environmental change on size-at-age in wild populations, in contrast to commonly reported effects of fishing. This finding has important implications for the management of fisheries as it indicates that a key trait associated with population productivity may be under considerably less short-term management control than currently assumed. Our study, overall, illustrates the need for a comparative approach within species for inferences concerning the many possible effects on body size of natural and anthropogenic drivers in the wild.

Relative prevalence of top-down versus bottom-up control in planktonic ecosystem under eutrophication and climate change: A comparative study of typical bay and estuary.

Eutrophication and climate change may affect the top-down versus bottom-up controls in aquatic ecosystems. However, the relative prevalence of the two controls in planktonic ecosystems along the eutrophication and climate gradients has rarely been addressed. Here, using the field surveys of 17 years in a typical bay and estuary, we test two opposite patterns of trophic control dominance and their response to regional temporal eutrophication and climate fluctuations. It was found that trophic control of planktonic ecosystems fluctuated between the dominance of top-down and bottom-up controls on time scales in both the bay and estuary studied. The relative prevalence of these two controls in both ecosystems was significantly driven directly by regional dissolved inorganic nitrogen but, for the estuary, also by the nonlinear effects of regional sea surface temperature. In terms of indirect pathways, community relationships (synchrony and grazing pressure) in the bay are driven by both regional dissolved inorganic nitrogen - soluble reactive phosphorus ratio and sea surface temperature, but this drive did not continue to be transmitted to the trophic control. Conversely, trophic control in estuary was directly related to grazing pressure and indirectly related to synchrony. These findings support the view that eutrophication and climate drive the relative prevalence of top-down versus bottom-up controls at ecosystem and temporal scales in planktonic ecosystems, which has important implications for predicting the potential impacts of anthropogenic and environmental perturbations on the structure and function of marine ecosystems.

Bottom-up production of injectable itraconazole suspensions using membrane technology.

Bottom-up production of active pharmaceutical ingredient (API) crystal suspensions offers advantages in surface property control and operational ease over top-down methods. However, downstream separation and concentration pose challenges. This proof-of-concept study explores membrane diafiltration as a comprehensive solution for downstream processing of API crystal suspensions produced via anti-solvent crystallization. It involves switching the residual solvent (N-methyl-2-pyrrolidone, NMP) with water, adjusting the excipient (d-α-Tocopherol polyethylene glycol 1000 succinate, TPGS) quantity, and enhancing API loading (solid concentration) in itraconazole crystal suspensions. NMP concentration was decreased from 9 wt% to below 0.05 wt% (in compliance with European Medicine Agency guidelines), while the TPGS concentration was decreased from 0.475 wt% to 0.07 wt%. This reduced the TPGS-to-itraconazole ratio from 1:2 to less than 1:50 and raised the itraconazole loading from 1 wt% to 35.6 wt%. Importantly, these changes did not adversely affect the itraconazole crystal stability in suspension. This study presents membrane diafiltration as a one-step solution to address downstream challenges in bottom-up API crystal suspension production. These findings contribute to optimizing pharmaceutical manufacturing processes and hold promise for advancing the development of long-acting API crystal suspensions via bottom-up production techniques at a commercial scale.

Bottom-Up Synthesized Glucan Materials: Opportunities from Applied Biocatalysis.

Linear d-glucans are natural polysaccharides of simple chemical structure. They are comprised of d-glucosyl units linked by a single type of glycosidic bond. Noncovalent interactions within, and between, the d-glucan chains give rise to a broad variety of macromolecular nanostructures that can assemble into crystalline-organized materials of tunable morphology. Structure design and functionalization of d-glucans for diverse material applications largely relies on top-down processing and chemical derivatization of naturally derived starting materials. The top-down approach encounters critical limitations in efficiency, selectivity, and flexibility. Bottom-up approaches of d-glucan synthesis offer different, and often more precise, ways of polymer structure control and provide means of functional diversification widely inaccessible to top-down routes of polysaccharide material processing. Here the natural and engineered enzymes (glycosyltransferases, glycoside hydrolases and phosphorylases, glycosynthases) for d-glucan polymerization are described and the use of applied biocatalysis for the bottom-up assembly of specific d-glucan structures is shown. Advanced material applications of the resulting polymeric products are further shown and their important role in the development of sustainable macromolecular materials in a bio-based circular economy is discussed.

A multiplex UPLC-MS/MS method for the quantification of three PD-L1 checkpoint inhibitors, atezolizumab, avelumab, and durvalumab, in human serum.

BACKGROUND AND AIM: To support pharmacokinetic studies, a multiplex UPLC-MS/MS assay was developed and validated to quantify PD-L1 checkpoint inhibitors atezolizumab, avelumab, and durvalumab in serum. METHODS: A bottom-up sample pre-treatment procedure was developed to determine atezolizumab, avelumab, and durvalumab in serum. This procedure consisted of (1) precipitation of the monoclonal antibody with ammonium sulfate, (2) reduction with dithiothreitol, (3) denaturation with methanol, and (4) tryptic digestion of the protein. The unique signature peptides resulting after sample pre-treatment of the antibodies were measured using UPLC-MS/MS with a total run time of 11 minutes. The clinical application was evaluated by analyzing 114 atezolizumab patient samples. RESULTS: The developed method was found to be accurate and precise for all three analytes over a concentration range of 3.00-150 µg/mL. No endogenous interference was present in serum samples. Cross-interference experiments showed no cross-analyte interference and acceptable cross-internal standard interference. In addition, no substantial carry-over was observed. The stable isotopically labeled signature peptides were most effective in compensating for matrix effects. Recovery based on back-calculated concentrations of calibration standards and quality control samples was found to be high. The analytes were stable for at least three freeze-thaw cycles, for 42 hours at processing conditions, for at least two days at 2-8°C in the final extract, for five days before re-injection analysis at 4°C, and long-term for at least 11 months at -70°C. The assay was tested for its applicability in clinical practice. For this purpose, 114 atezolizumab patient samples were measured. CONCLUSION: A multiplex UPLC-MS/MS assay was developed and validated to quantify atezolizumab, avelumab, and durvalumab in human serum. The applicability of this method was demonstrated by the analysis of clinical atezolizumab samples. The method is suitable to support clinical pharmacokinetic studies involving atezolizumab, avelumab, or durvalumab.

Amino-functional magnetic covalent organic framework as an effective adsorbent for the determination of neonicotinoids in food samples.

Recently, covalent organic frameworks have gained popularity in sample pretreatment. However, the application of covalent organic frameworks in the enrichment of hydrophilic compounds remains a challenge. Thus, a functionalized magnetic covalent organic framework equipped with amino groups was constructed using a bottom-up functionalization strategy. Considering the advantages of this novel adsorbent such as high porosity, large adsorption capacity, and hydrophilic surface, a sensitive magnetic solid-phase extraction coupled with high-performance liquid chromatography-tandem mass spectrometry method was established for the effective determination of neonicotinoids. This method exhibited good linearities with correlation coefficients ranging from 0.9983 to 0.9995, low detection limits in the range 0.003-0.009 ng g-1 and 0.001-0.013 ng mL-1, and limits of quantification in the range 0.010-0.031 ng g-1 and 0.004-0.044 ng mL-1. Furthermore, satisfactory repeatability with relative standard deviations ≤ 6.7% and spiked recoveries between 82.3 and 99.8% were obtained. This work not only provided a promising adsorbent for the sensitive determination of trace-level neonicotinoids but also represented a unique insight for effective enrichment of super hydrophilic hazards.

Accelerating Proteomics Using Broad Specificity Proteases.

Trypsin is the gold-standard protease in bottom-up proteomics, but many sequence stretches of the proteome are inaccessible to trypsin and standard LC-MS approaches. Thus, multienzyme strategies are used to maximize sequence coverage in post-translational modification profiling. We present fast and robust SP3- and STRAP-based protocols for the broad-specificity proteases subtilisin, proteinase K, and thermolysin. All three enzymes are remarkably fast, producing near-complete digests in 1-5 min, and cost 200-1000× less than proteomics-grade trypsin. Using FragPipe resolved a major challenge by drastically reducing the duration of the required "unspecific" searches. In-depth analyses of proteinase K, subtilisin, and thermolysin Jurkat digests identified 7374, 8178, and 8753 unique proteins with average sequence coverages of 21, 29, and 37%, including 10,000s of amino acids not reported in PeptideAtlas' >2400 experiments. While we could not identify distinct cleavage patterns, machine learning could distinguish true protease products from random cleavages, potentially enabling the prediction of cleavage products. Finally, proteinase K, subtilisin, and thermolysin enabled label-free quantitation of 3111, 3659, and 4196 unique Jurkat proteins, which in our hands is comparable to trypsin. Our data demonstrate that broad-specificity proteases enable quantitative proteomics of uncharted areas of the proteome. Their fast kinetics may allow "on-the-fly" digestion of samples in the future.

Fit for Purpose Approach To Evaluate Detection of Amino Acid Substitutions in Shotgun Proteomics.

Amino acid substitutions (AASs) alter proteins from their genome-expected sequences. Accumulation of substitutions in proteins underlies numerous diseases and antibiotic mechanisms. Accurate global detection of AASs and their frequencies is crucial for understanding these mechanisms. Shotgun proteomics provides an untargeted method for measuring AASs but introduces biases when extrapolating from the genome to identify AASs. To characterize these biases, we created a "ground-truth" approach using the similarities betweenEscherichia coli and Salmonella typhimurium to model the complexity of AAS detection. Shotgun proteomics on mixed lysates generated libraries representing ∼100,000 peptide-spectra and 4161 peptide sequences with a single AAS and defined stoichiometry. Identifying S. typhimurium peptide-spectra with only the E. coli genome resulted in 64.1% correctly identified library peptides. Specific AASs exhibit variable identification efficiencies. There was no inherent bias from the stoichiometry of the substitutions. Short peptides and AASs localized near peptide termini had poor identification efficiency. We identify a new class of "scissor substitutions" that gain or lose protease cleavage sites. Scissor substitutions also had poor identification efficiency. This ground-truth AAS library reveals various sources of bias, which will guide the application of shotgun proteomics to validate AAS hypotheses.

Testing effects of bottom-up factors, grazing, and competition on New Zealand rocky intertidal algal communities.

Top-down and bottom-up factors and their interaction highlight the interdependence of resources and consumer impacts on food webs and ecosystems. Variation in the strength of upwelling-mediated ecological controls (i.e., light availability and herbivory) between early and late succession stages is less well understood from the standpoint of influencing algal functional group composition. We experimentally tested the effect of light, grazing, and disturbance on rocky intertidal turf-forming algal communities. Studies were conducted on the South Island of New Zealand at Raramai on the east coast (a persistent downwelling region) and Twelve Mile Beach on the west coast (an intermittent upwelling region). Herbivory, light availability, and algal cover were manipulated and percent cover of major macroalgal functional groups and sessile invertebrates were measured monthly from October 2017 to March 2018. By distinguishing between algal functional groups and including different starting conditions in our design, we found that the mosaic-like pattern of bare rock intermingled with diverse turf-forming algae at Twelve Mile Beach was driven by a complex array of species interactions, including grazing, predation, preemptive competition and interference competition, colonization rates, and these interactions were modulated by light availability and other environmental conditions. Raramai results contrasted with those at Twelve Mile Beach in showing stronger effects of grazing and relatively weak effects of other interactions, low colonization rates of invertebrates, and light effects limited to crustose algae. Our study highlights the potential importance of an upwelling-mediated 3-way interaction among herbivory, light availability, and preemption in structuring contrasting low rocky intertidal macroalgal communities.

Collective Cell Radial Ordered Migration in Spatial Confinement.

Collective cells, a typical active matter system, exhibit complex coordinated behaviors fundamental for various developmental and physiological processes. The present work discovers a collective radial ordered migration behavior of NIH3T3 fibroblasts that depends on persistent top-down regulation with 2D spatial confinement. Remarkably, individual cells move in a weak-oriented, diffusive-like rather than strong-oriented ballistic manner. Despite this, the collective movement is spatiotemporal heterogeneous and radial ordering at supracellular scale, manifesting as a radial ordered wavefront originated from the boundary and propagated toward the center of pattern. Combining bottom-up cell-to-extracellular matrix (ECM) interaction strategy, numerical simulations based on a developed mechanical model well reproduce and explain above observations. The model further predicts the independence of geometric features on this ordering behavior, which is validated by experiments. These results together indicate such radial ordered collective migration is ascribed to the couple of top-down regulation with spatial restriction and bottom-up cellular endogenous nature.