Tracking of lineage mass via quantitative phase imaging and confinement in low refractive index microwells.

Measurements of cell lineages are central to a variety of fundamental biological questions, ranging from developmental to cancer biology. However, accurate lineage tracing requires nearly perfect cell tracking, which can be challenging due to cell motion during imaging. Here we demonstrate the integration of microfabrication, imaging, and image processing approaches to demonstrate a platform for cell lineage tracing. We use quantitative phase imaging (QPI), a label-free imaging approach that quantifies cell mass. This gives an additional parameter, cell mass, that can be used to improve tracking accuracy. We confine lineages within microwells fabricated to reduce cell adhesion to sidewalls made of a low refractive index polymer. This also allows the microwells themselves to serve as references for QPI, enabling measurement of cell mass even in confluent microwells. We demonstrate application of this approach to immortalized adherent and nonadherent cell lines as well as stimulated primary B cells cultured ex vivo. Overall, our approach enables lineage tracking, or measurement of lineage mass, in a platform that can be customized to varied cell types.

Direct observation correlates NFκB cRel in B cells with activating and terminating their proliferative program.

Antibody responses require the proliferative expansion of B cells controlled by affinity-dependent signals. Yet, proliferative bursts are heterogeneous, varying between 0 and 8 divisions in response to the same stimulus. NFκB cRel is activated in response to immune stimulation in B cells and is genetically required for proliferation. Here, we asked whether proliferative heterogeneity is controlled by natural variations in cRel abundance. We developed a fluorescent reporter mTFP1-cRel for the direct observation of cRel in live proliferating B cells. We found that cRel is heterogeneously distributed among naïve B cells, which are enriched for high expressors in a heavy-tailed distribution. We found that high cRel expressors show faster activation of the proliferative program, but do not sustain it well, with population expansion decaying earlier. With a mathematical model of the molecular network, we showed that cRel heterogeneity arises from balancing positive feedback by autoregulation and negative feedback by its inhibitor IκBε, confirmed by mouse knockouts. Using live-cell fluorescence microscopy, we showed that increased cRel primes B cells for early proliferation via higher basal expression of the cell cycle driver cMyc. However, peak cMyc induction amplitude is constrained by incoherent feedforward regulation, decoding the fold change of cRel activity to terminate the proliferative burst. This results in a complex nonlinear, nonmonotonic relationship between cRel expression and the extent of proliferation. These findings emphasize the importance of direct observational studies to complement gene knockout results and to learn about quantitative relationships between biological processes and their key regulators in the context of natural variations.

Considerations for fatty acids in standardized reference diet for parthenogenetic marbled crayfish Procambarus virginalis model organism.

Fatty acid accumulation was studied in the parthenogenetic all-female marbled crayfish Procambarus virginalis using six arbitrarily designed experimental feeds and related to individuals with glair glands (sexual maturity) after 100 days of ad libitum feeding at 21 °C, including gravid females from the wild as a reference. Fatty acids 16:0 and 18:1n-9 comprised 40% of the total amount of fatty acids and tended to up-concentrate in bodies. Shorter chain 14:0 depleted from feed to body. Across diets, there was a concomitant decrease in precursor fatty acid and increase in product fatty acid, such as reinforcements in monounsaturated fatty acid (18:1n-9), eicosanoid precursors 20:4n-6 (arachidonic acid, ARA) and 20:5n-3 (eicosapentaenoic acid, EPA) in-vivo, but not 22:6n-3 (docosahexaenoic acid, DHA) except when deficient in CHI or CHI + SPI diets. Saturation kinetics modeling (R2 0.7-0.9, p < 0.05) showed that when the ARA share is ~ 1%, the EPA share is ~ 8%, and the DHA share is ~ 2% in the food lipids, the accumulation of fatty acids in body lipids levels off. The lowest DHA in the CHI (0% glair glands) or CHI + SPI (0-3.9% glair glands) diets, and the lowest ARA in SER (0% glair glands) or SER + SPI (0-3% glair glands) diets, were synchronous with negligible sexual maturity despite a wide range of observed specific growth rates (2.77-3.60% per day), body size (0.44-0.84 g), ≤ 5% crude lipid and 40-46% crude protein feed. The FISH and SHRIMP diets (56% protein, 11-14% lipid) with the highest ARA, EPA, and DHA together seem to be the most conducive diets for sexual maturity (up to 20% of individuals with glair glands). We propose a fatty acid profile mimicking the FISH or SHRIMP diets as a starting point for designing the lipid content required in the marbled crayfish standardized reference diet.

Considerations for protein and amino acids in standardized reference diet for parthenogenetic marbled crayfish Procambarus virginalis model organism.

The concept of a standardized reference diet (SRD) is used in laboratory model organisms to ensure nutritional control between studies and laboratories. Although models using the genetically identical, all female parthenogenetic marbled crayfish (Procambarus virginalis) are growing in popularity, research into nutrition in this species still has many knowledge gaps. To fast track the development of a SRD in terms of protein and amino acids (SRDprotein) for this species, we first analyzed the composition of its body amino acids to determine the ideal protein concept (IPC) of indispensable amino acids in wild-caught P. virginalis (which had an unusually high preponderance of leucine and arginine). Then, we strategically evaluated three common clusters of types of fish feed: (1) ornamental fish feed (SER) fortified with a naturally occurring alga (Spirulina). This type of feed was protein-high in arginine and leucine (SER + SPI) that fulfils the species' IPC for iso-protein (~ 40%), iso-phosphorus (~ 0.8%) and near iso-energetic (~ 475 kcal 100 g-1); (2) freeze-dried live feed consisting of chironomid larvae (CHI) fortified with Spirulina (CHI + SPI) that fulfils the IPC for iso-protein (~ 46%), iso-phosphorus (~ 0.7%) and near iso-energetic (~ 405 kcal 100 g-1); and (3) a commercially standardized 'starter diet' for carnivorous fish larvae (FISH) and post-larval shrimps (SHRIMP) with iso-protein (~ 56%) and iso-phosphorus (~ 1.6%). A total of six diets, embracing a diverse range of proteinaceous feeds, were used in a 100-day ad libitum feeding and growth trial. The FISH group outperformed all the other groups (p < 0.05) and our exploratory multivariate analysis revealed an ideal demand of > 44% protein (tailored to deliver high arginine 3% and leucine 4%, followed by the usual lysine > 3.5% and methionine 1.2%) but also the lowest carbohydrate level (21%). For SRDprotein, our findings show that the FISH diet is ideal and suggest the possibilities of using a CHI + SPI diet for further optimization (more economic use of protein and phosphorus).

Fish stocks as phosphorus sources or sinks: Influenced by nutritional and metabolic variations, not solely by dietary content and stoichiometry.

The study provides a descriptive understanding of when fish (Cyprinus carpio model) are the source or sink of phosphorus. Dissolved reactive phosphorus (DRP; PO4-P) losses (51.1 ± 5.9 % of intake-P) increase at excess of bioavailable P (>0.83 g 100 g-1 dry matter, DM fed) or when food (digestible) N:P mass ratio (≤4.4:1) approaches organismal storage threshold (~4:1). This is known, however, even at a sub-threshold food P content (0.57 g 100 g-1 DM) and food N:P mass ratio (7.3:1), DRP losses (57.8 ± 4.5 % of intake-P) may be extraordinary if two indispensable amino acids are biologically insufficient (lysine ≤1.43 g, methionine ≤0.39 g 100 g-1 DM fed). Given that methionine and lysine are sufficient, DRP losses cease (≈0 %) and even some P from water is absorbed, given there is support from non-protein energy (NPE). Insufficient NPE (<180 kcal 100 g-1 DM fed) may drive DRP losses (81.6 ± 4.3 % of intake-P) beyond expected levels (46-59 % of intake-P) at a given food P content (0.91 g 100 g-1 DM). Natural food seldom fulfills low P, high lysine + methionine, and high NPE contents simultaneously, thus keeping fish in a perpetual P recycling for algae (scaleless carp > scaly carp). Such P recycling ceases only during basal metabolism. During feeding state, the richness of lysine + methionine bound N and lipid + carbohydrate bound C in the food base may enhance the fishes' threshold of P storage. P storage can be diminished when they are insufficient. We show that for fish, the decision of P recycling or not recycling (for algae) may change based on the supply of specific fractions of N or C from the food web or metabolic variations (basal metabolism, presence of scales). NOVELTY STATEMENT: The ecological stoichiometry theory is better connected to fish nutritional bioenergetics for better understanding and biomanipulation of eutrophication processes.

Synergistic digestibility effect by planktonic natural food and habitat renders high digestion efficiency in agastric aquatic consumers.

A digestibility enhancing effect of natural food on stomachless fish model (Cyprinus carpio) was verified by fluorogenic substrate assays of enzymatic activities in experimental pond carp gut flush and planktonic food over a full vegetative season. Then compared with size-matched conspecific grown artificially (tank carp) and an advanced omnivore species possessing true stomach (tilapia, Oreochromis niloticus). Results suggested activities of digestive enzymes (except amylolytic) were significantly higher in pond carp (p ≤ 0.05) than in the size-matched tank carp. Even compared to tilapia, pond carp appeared superior (p < 0.05; proteolytic or chitinolytic activities) or comparable (p > 0.05; phosphatase or cellulolytic activities). Amylolytic, chitinolytic, and phosphatases activities in pond carp gut significantly increased (p ≤ 0.01) over season. Several orders-of-magnitude higher enzymatic activities were detected in planktonic natural food than expressed in carp gut. Amino acid markers in planktonic food revealed a higher share of zooplankton (microcrustaceans), but not phytoplankton, synchronized with higher activities of complex polysaccharide-splitting enzymes (cellulolytic and chitinolytic) in fish gut. Periods of clear water phase low in chlorophyll-a and nutrients, but high in certain zooplankton (preferably cladocerans), may create a synergistic digestibility effect in pond carp. We conclude aquatic ecosystem components (natural food, water, microbiota) enhance fishes' hydrolyzing capabilities of C/N/P macromolecules and even their complex polymers such as cellulose, chitin, and maybe phytate (to be validated), to the extent that being stomachless is not an issue. Aquatic nutritional ecologists may consider that laboratory-based understandings of digestibility may underestimate digestion efficiency of free-ranging fish in ponds or lakes.

Nutrient footprint versus EPA + DHA security in land-locked regions-more of local pond farmed, imported marine fish or fish oil capsules?

EPA + DHA intake in land-locked central Europe (CE) is barely fulfilled. Imported marine fish/farmed salmonids are likely the backbone of an ailing EPA + DHA security. Supplementing with captured marine fish oil capsules (~0.5 g up to 1.6 g CO2-eq. mg EPA + DHA-1) could be comparable in GHG emissions with fish consumption itself (~1 g to as low as 0.6 g CO2-eq. mg EPA + DHA-1). But synergistic benefits of EPA + DHA intake by consuming fish protein need consideration too. Taking semi-intensive pond carp and intensively farmed salmon as models, we analyzed footprint, eco-services, and resource use efficiency perspectives of achieving EPA + DHA security in a CE region. Despite a lower production footprint, pond-farmed fish greatly lag in EPA + DHA supply (carp 101-181 mg 100 g-1 < salmon 750-1300 mg 100 g-1). It doubles-to-quadruples footprint 'per mg' of EPA + DHA: nitrogen (carp 18.3 > salmon 8.7 mg N), phosphorus (carp 6.8 > salmon 1.6 mg P), and climate change (carp 1.84 > salmon 0.8 g CO2-eq.). With enhancements in pond carp (>300 mg EPA + DHA 100 g-1), these differences may cease to exist. Harnessing EPA + DHA bioaccumulation pathways active in ponds, finishing feeding strategies, and polyculture, the EPA + DHA content in pond fish may be increased. Ecosystem services with EPA + DHA mining from pond food web or high EPA + DHA output-to-input ratio (pond carp 1-200 > RAS salmon 0.75) make ponds an eco-efficient system. As fish consumption in CE must improve, pond-farmed fish would be needed to complement (but not substitute) salmonid/marine fish/oil capsules consumption. Achieving EPA + DHA security with minimum pressure on the environment or global resources.

Protein from whole-body crayfish homogenate may be a high supplier of leucine or branched-chain amino acids - A call for validation on genus Procambarus sp.

Essential proteinogenic branched-chain amino acids (BCAA), particularly leucine (Leu) have been investigated for their role in enhancing human myofibrillar protein synthesis and biomedical research on tumor models. However, only a few protein sources in our current food system have high enough BCAA or Leu coefficients (% of total amino acids) to be considered as supplements for food, sport, or biomedical research. Mostly dairy-sourced proteins such as casein and whey or rarely plant source such as maize gluten are typically regarded as the gold standards. This study hypothesized that protein isolates derived from the whole-body homogenate (including the chitinous exoskeleton) of procambarid crayfish might exhibit unusually high BCAA and Leu content. The study provides open-access data on the amino acid compositions of two procambarid crayfish (Procambarus virginalis and P. clarkii), as well as a comparison with casein. The mentioned crayfish species could offer 6.36-7.39 g Leu 100 g-1 dry matter (at 43-48% protein only). Crayfish whole-body protein isolates exhibit a Leu coefficient (18.41±2.51% of total amino acids) and a BCAA coefficient (28.76±2.39% of total amino acids), which is comparable to or higher than of casein (Leu coefficient 8.65±0.08%; BCAA coefficient 20.03±0.73%). However, it is important to interpret these results with caution, due to the challenges associated with leucine and isoleucine separation, as well as potential interactions within the sample matrices. Hence, international validation of these findings is recommended. NOVELTY STATEMENT: Protein isolates from whole-body homogenate (including chitinous exoskeleton) of P. virginalis and/or P. clarkii are hypothesized to be dense in BCAA and Leu. For potential use in biomedical research or as additives in supplements for BCAA and Leu.

Mechanistic insight into the protective and pathogenic immune-responses against SARS-CoV-2.

COVID-19 is a severe respiratory illness that has emerged as a devasting health problem worldwide. The disease outcome is heterogeneous, which is most likely dependent on the immunity of an individual. Asymptomatic and mildly/moderate symptomatic (non-severe) patients likely develop an effective early immune response and clear the virus. However, severe symptoms dominate due to a failure in the generation of an effective and specific early immune response against SARS-CoV-2. Moreover, a late surge in pathogenic inflammation involves dysregulated innate and adaptive immune responses leading to local and systemic tissue damage and the emergence of severe disease symptoms. In this review, we describe the potential mechanisms of protective and pathogenic immune responses in "mild/moderate" and "severe" symptomatic SARS-CoV-2 infected people, respectively, and discuss the immune components that are currently targeted for therapeutic intervention.

The NFκB signaling system in the generation of B-cell subsets: from germinal center B cells to memory B cells and plasma cells.

Memory B cells and antibody-secreting cells are the two prime effector B cell populations that drive infection- and vaccine-induced long-term antibody-mediated immunity. The antibody-mediated immunity mostly relies on the formation of specialized structures within secondary lymphoid organs, called germinal centers (GCs), that facilitate the interactions between B cells, T cells, and antigen-presenting cells. Antigen-activated B cells may proliferate and differentiate into GC-independent plasmablasts and memory B cells or differentiate into GC B cells. The GC B cells undergo proliferation coupled to somatic hypermutation of their immunoglobulin genes for antibody affinity maturation. Subsequently, affinity mature GC B cells differentiate into GC-dependent plasma cells and memory B cells. Here, we review how the NFκB signaling system controls B cell proliferation and the generation of GC B cells, plasmablasts/plasma cells, and memory B cells. We also identify and discuss some important unanswered questions in this connection.

Single-sequence protein structure prediction using a language model and deep learning.

AlphaFold2 and related computational systems predict protein structure using deep learning and co-evolutionary relationships encoded in multiple sequence alignments (MSAs). Despite high prediction accuracy achieved by these systems, challenges remain in (1) prediction of orphan and rapidly evolving proteins for which an MSA cannot be generated; (2) rapid exploration of designed structures; and (3) understanding the rules governing spontaneous polypeptide folding in solution. Here we report development of an end-to-end differentiable recurrent geometric network (RGN) that uses a protein language model (AminoBERT) to learn latent structural information from unaligned proteins. A linked geometric module compactly represents Cα backbone geometry in a translationally and rotationally invariant way. On average, RGN2 outperforms AlphaFold2 and RoseTTAFold on orphan proteins and classes of designed proteins while achieving up to a 106-fold reduction in compute time. These findings demonstrate the practical and theoretical strengths of protein language models relative to MSAs in structure prediction.

Regulation of T-independent B-cell responses by microRNA-146a.

The microRNA, miR-146a, is a negative feedback regulator of the central immune transcription factor, nuclear factor kappa B (NFkB). MiR-146a plays important roles in the immune system, and miR-146a deficient mice show a complex phenotype with features of chronic inflammation and autoimmune disease. In this study, we examined the role of miR-146a in extrafollicular B-cell responses, finding that miR-146a suppresses cellular responses in vivo and in vitro. Gene expression profiling revealed that miR-146a-deficient B-cells showed upregulation of interferon pathway genes, including Traf6, a known miR-146a target. We next interrogated the role of TRAF6 in these B-cell responses, finding that TRAF6 is required for proliferation by genetic and pharmacologic inhibition. Together, our findings demonstrate a novel role for miR-146a and TRAF6 in the extrafollicular B-cell responses, which have recently been tied to autoimmune disease pathogenesis. Our work highlights the pathogenetic role of miR-146a and the potential of pharmacologic inhibition of TRAF6 in autoimmune diseases in which miR-146a is deregulated.

TILAFeed: A bio-based inventory for circular nutrients management and achieving bioeconomy in future aquaponics.

Future food systems aim to achieve improved resource use efficiency and minimized environmental footprint through a circular bioeconomy-based approach. Aquaponics is a hallmark of such circular food production. The image of a circular nutrient utilization efficiency in aquaponics is often weakened by the daily use of additional inorganic fertilizers in such systems. As circular bioeconomy greatly emphasizes developing bio-based solutions, the presented novel inventory 'TilaFeed' and its associated utility tools is a step towards achieving more circular nutrient utilization and bioeconomy in future aquaponics. Through the formulation of tailored fish feed that is compatible with aquaponic systems' needs (e.g. plant nutrient requirement, mineralization efficiency of microbial sludge digesters), the objectives of TilaFeed are (i) to solve nutrient constraints in aquaponic systems, both for fish and plants; (ii) to avoid or strongly limit artificial fertilizer use in aquaponics by smartly tailored aquafeeds; and (iii) to equip system managers with decision-making tools for improved nutrient planning of their aquaponic systems. TilaFeed is a bio-based inventory. It integrates material (nutrient) flow information from feed to fish (in-vivo nutrient partitioning, forms of excretion) to environment (in-situ nutrient loading, nutrient forms) and primary producers (mineralization by microbes, available nutrients to plants). Based on TilaFeed-Model, feed for future aquaponics may be more precisely formulated with the principle that nutrients are not only a resource for fish, but excreted nutrients from fish (feed) also fertilize the microbes and plants.

Starter feed for carnivorous species as a practical replacement of bloodworms for a vertebrate model organism in ageing, the turquoise killifish Nothobranchius furzeri.

The absence of a controlled diet is unfortunate in a promising model organism for ageing, the turquoise killifish (Nothobranchius furzeri Jubb, 1971). Currently captive N. furzeri are fed bloodworms but it is not known whether this is an optimal diet. Replacing bloodworms with a practical dry feed would reduce diet variability. In the present study, we estimated the nutritional value of the diet ingested by wild fish and determined the fish-body amino acid profile as a proxy for their nutritional requirements. We compared the performance of fish fed four commercial feeds containing 46%-64% protein to that achieved with bloodworms and that of wild fish. Wild fish target a high-protein (60%) diet and this is supported by their superior performance on high-protein diets in captivity. In contrast, feeds for omnivores led to slower growth, lower fecundity and unnatural liver size. In comparison to wild fish, a bloodworm diet led to lower body condition, overfeeding and male liver enlargement. Out of the four dry feeds tested, the fish fed Aller matched wild fish in body condition and liver size, and was comparable to bloodworms in terms of growth and fecundity. A starter feed for carnivorous species appears to be a practical replacement for bloodworms for N. furzeri. The use of dry feeds improved performance in comparison to bloodworms and thus may contribute to reducing response variability and improving research reproducibility in N. furzeri research.

Icy affairs: Understanding recent advancements in the freezing and frozen storage of fish.

Freezing methods have evolved over the last 30 years. This review states the effect of various freezing methods on the quality of fish and seafood. Freezing temperatures, freezing, and frozen storage temperatures were also analyzed and reviewed. The changes in the ice crystal, protein, and lipid affect the fish quality and nutritional value during freezing and frozen storage. Freezing methods when combined with various additives or preprocessing approaches help improve the efficacy of freezing and frozen storage. Several experimental or emerging methods also have positive effects on the products' quality. According to the metadata reanalysis of quality markers, freshly frozen fish using different freezing methods may vary much in terms of ice diameter, but not others. High pressure freezing or immersion freezing-derived fish retains the best quality through frozen storage. More data are required on freezing methods (electrical-assisted freezing, microwave-assisted freezing, magnetic-assisted freezing, radiofrequency-assisted freezing, and the commercial's application and investment should be considered in the future. This review sheds light on finding a balanced initial shear force during freezing and the use of certain additives to control freezing-related damages. Focusing on ice diameter alone may be futile (e.g., liquid N2 freezing). Future optimization of technologies should be in a way that several processes along the farm to fork such as freezing, frozen storage, thawing, thermal processing of fish, and even refabrication of food should mutually complement each other's needs to deliver safe and high-quality fish to the consumer's plate, even after a prolonged shelf-life.

Understanding Nutrition and Metabolism of Threatened, Data-Poor Rheophilic Fishes in Context of Riverine Stocking Success- Barbel as a Model for Major European Drainages?

Large-bodied, river-migrating, rheophilic fishes (cyprinids) such as barbel Barbus barbus, nase Chondrostoma nasus, asp Leuciscus aspius, and vimba bream Vimba vimba are threatened in major European drainages. This represents the subject of our present study. Their hatchery nutrition prior to river-release is mostly on a hit-and-trial or carp-based diet basis. The study demonstrates an alternative approach to decide optimum nutrition for these conservation-priority and nutritionally data-poor fishes. The study revealed barbel as a central representative species in terms of wild body composition among other native rheophilic cyprinids considered (asp, nase, vimba bream). Taking barbel as a model, the study shows that barbel or rheophilic cyprinids may have carnivorous-like metabolism and higher requirements of S-containing, aromatic, branched-chain amino acids (AAs) than carps. Besides, there are important interactions of AAs and fatty acids (FAs) biosynthesis to consider. Only proper feeding of nutritionally well-selected diets may contribute to river stocking mandates such as steepest growth trajectory (≈less time in captivity), ideal size-at-release, body fitness (≈blend-in with wild conspecifics, predator refuge), better gastrointestinal condition, maximized body reserves of functional nutrients, and retention efficiencies (≈uncompromised physiology). Considering important physiological functions and how AA-FA interactions shape them, hatchery-raised fishes on casually chosen diets may have high chances of physiological, morphological, and behavioral deficits (≈low post-stocking survivability). Based on the observations, optimum nutrient requirements of juvenile (0+ to 1+ age) barbels are suggested. Future efforts may consider barbels as a nutrition model for conservation aquaculture of threatened and data poor rheophilic cyprinids of the region.

Acceleration time history dataset for a 3D miniature model of a shear building with structural damage.

The dynamics of short-to-medium height frame buildings closely resembles with a shear building. The vibrational response from a shear building model will help to understand and carry out a detailed study over the behaviour of these buildings. This data article provides acceleration time history response from a laboratory-based small-scaled (scale 1:20) three-dimensional six-storey shear building model. The article presents model specification, its assembly and experimental set-up for acquiring these data. The dataset contains free vibrational responses from the structure under both undamaged and damaged conditions. This laboratory model and corresponding experimental data are a valuable asset for the researchers working on analytical and numerical model generation, particularly in the field of structural health monitoring, in order to experimentally verify their newly developed methodologies for damage identification. The authors have provided this data to assist and contribute so as to benefit the state-of-the-art. All the datasets can be accessed via the repository link provided in the specification table. The article also provides a complete model assembly and experimentation video to its readers for a better insight into the data acquisition steps.

A mimotope attached to an ITIM-SHP-1 interaction inhibitory peptide boosts immune response and efficacy.

Antigen binding to the B-cell receptor initiates a downstream signalling pathway that contains both stimulatory and damping components. A malarial parasite-derived conformation-constrained peptide was conjugated to a signal-damping pathway inhibitor. Mice immunized with this antigen produced higher antibody levels which delayed parasitemia. This represents a new approach to antigen design.

Bi-FPNFAS: Bi-Directional Feature Pyramid Network for Pixel-Wise Face Anti-Spoofing by Leveraging Fourier Spectra.

The emergence of biometric-based authentication using modern sensors on electronic devices has led to an escalated use of face recognition technologies. While these technologies may seem intriguing, they are accompanied by numerous implicit drawbacks. In this paper, we look into the problem of face anti-spoofing (FAS) on a frame level in an attempt to ameliorate the risks of face-spoofed attacks in biometric authentication processes. We employed a bi-directional feature pyramid network (BiFPN) that is used for convolutional multi-scaled feature extraction on the EfficientDet detection architecture, which is novel to the task of FAS. We further use these convolutional multi-scaled features in order to perform deep pixel-wise supervision. For all of our experiments, we performed evaluations across all major datasets and attained competitive results for the majority of the cases. Additionally, we showed that introducing an auxiliary self-supervision branch tasked with reconstructing the inputs in the frequency domain demonstrates an average classification error rate (ACER) of 2.92% on Protocol IV of the OULU-NPU dataset, which is significantly better than the currently available published works on pixel-wise face anti-spoofing. Moreover, following the procedures of prior works, we performed inter-dataset testing, which further consolidated the generalizability of the proposed models, as they showed optimum results across various sensors without any fine-tuning procedures.