bluuebunny/crossref_metadata_2025_split huggingface dataset:
Detection of DNA replication errors and 8-oxo-dGTP-mediated mutations inE. coliby Duplex DNA Sequencing
Authors: Bhawsinghka Niketa , Burkholder Adam , Schaaper Roel M. 
Published: October 2022

AbstractMutation is a phenomenon inescapable for all life-forms, including bacteria. While bacterial mutation rates are generally low due to the operation of error-avoidance systems, sometimes they are elevated by many orders of magnitude. Such a state, known as a hypermutable state, can result from exposure to stress or to harmful environments. Studies of bacterial mutation frequencies and analysis of the precise types of mutations can provide insights into the mechanisms by which mutations occur and the possible involvement of error-avoidance pathways. Several approaches have been used for this, like reporter assays involving non-essential genes or mutation accumulation over multiple generations. However, these approaches give an indirect estimation, and a more direct approach for determining mutations is desirable. With the recent development of a DNA sequencing technique known as Duplex Sequencing, it is possible to detect rare variants in a population at a frequency of 1 in 107base pairs or less. Here, we have applied Duplex Sequencing to study spontaneous mutations inE. coli. We also investigated the production of replication errors by using a mismatch-repair defective (mutL) strain as well as oxidative-stress associated mutations using amutT-defective strain. For DNA from a wild-type strain we obtained mutant frequencies in the range of 10-7to 10-8depending on the specific base-pair substitution, but we argue that these mutants merely represent a background of the system, rather than mutations that occurredin vivo. In contrast, bona-fidein vivomutations were identified for DNA from both themutLandmutTstrains, as indicated by specific increases in base substitutions that are fully consistent with their establishedin vivoroles. Notably, the data reproduce the specific context effects ofin vivomutations as well as the leading vs. lagging strand bias among DNA replication errors.
Somatotopic organization of mechanosensory afferents in the stellate ganglion of the squid,Euprymna
Authors: Crook Robyn J 
Published: October 2022

AbstractCephalopod molluscs are growing in popularity and use as comparative models of complex brains and behaviors. Although the gross anatomy of their central and peripheral nervous systems have been well characterized for decades, there is still very limited information about the diversity of cell types in each ganglion or lobe, their arrangement or their network properties. Unlike more standard neuroscience models, there are limited tools available for cephalopods and few validated techniques for imaging neural activity. Here, live calcium imaging in a reduced preparation of the stellate ganglion and mantle tissue reveals mechanosensory afferents and interneurons, which are arranged somatotopically in the ganglion. Retrograde labeling from stellate nerves confirms that neurons sending axonal projections to distinct dermatomes are organized in roughly oblong clusters along the dorsal side of the ganglion. This is the first demonstration of afferent somatotopy in cephalopods, and the first direct visualization of mechanoreceptive and mechano-nociceptive neurons that fire in response to localized, firm touch on the body surface. The methods and findings in this study open multiple new lines of enquiry related to sensory processing in the cephalopod nervous system.
Close encounters of three kinds: impacts of leg, wing, and body collisions on flight performance in carpenter bees
Authors: Burnett Nicholas P. , Combes Stacey A. 
Published: October 2022

AbstractFlying insects often forage among cluttered vegetation that forms a series of obstacles in their flight path. Recent studies have focused on behaviors needed to navigate clutter while avoiding all physical contact, and as a result, we know little about flight behaviors that do involve encounters with obstacles. Here, we challenged carpenter bees (Xylocopa varipuncta) to fly through narrow gaps in an obstacle course to determine the kinds of obstacle encounters they experience, as well as the consequences for flight performance. We observed three kinds of encounters: leg, body, and wing collisions. Wing collisions occurred most frequently (in about 40% of flights, up to 25 times per flight) but these had little effect on flight speed or body orientation. In contrast, body and leg collisions, which each occurred in about 20% of flights (1-2 times per flight), resulted in decreased flight speeds and increased rates of body rotation (yaw). Wing and body collisions, but not leg collisions, were more likely to occur in wind versus still air. Thus, physical encounters with obstacles may be a frequent occurrence for insects flying in some environments, and the immediate effects of these encounters on flight performance depends on the body part involved.
Incidental eagle carcass detection can contribute to fatality estimation at operating wind facilities
Authors: Hallingstad Eric , Riser-Espinoza Daniel , Brown Samantha 
Published: October 2022

AbstractRisk of birds colliding with wind turbines, especially protected species like bald eagle and golden eagle, is a fundamental wildlife challenge the wind industry faces when developing and operating projects. The U.S. Fish and Wildlife Service requires wind facilities that obtain eagle take permits to document permit compliance through eagle fatality monitoring. If trained Operations and Maintenance (O&M) staff can reliably detect and report carcasses during their normal routines, then their ‘incidental detections’ could contribute substantially to meeting monitoring requirements required by eagle take permits. The primary objective of this study was to quantify incidental detection of eagle carcasses by O&M staff under a variety of landscape contexts and environmental conditions throughout 1 year. We used the incidental detection probabilities (proportion of decoys detected by O&M staff), along with raptor carcass persistence data and area adjustments, to calculate overall probability of incidental detection (i.e., incidentalg). We used feathered turkey decoys as eagle-carcass surrogates for monthly detection trials at 6 study sites throughout the U.S. We evaluated the primary drivers of incidental detection using logit regression models including season, viewshed complexity, and a derived variable called the “density quartile” as covariates. We used an Evidence of Absence-based approach to estimate the overall probability of incidental detection. Detection probabilities decreased as viewshed complexity increased and as distance from the turbine increased. The resulting overall probability of incidental detection for the 12-month period ranged from 0.07 to 0.47 (mean = 0.31). The primary drivers of variability in incidentalgwere detection probability and the area adjustment. Results of our research show that O&M staff were capable of incidentally detecting trial carcasses while performing their typical duties. Incorporating incidental detection by O&M staff in eagle fatality monitoring efforts is a reliable means of improving estimates of a facility’s direct impacts on eagles.
Missense and nonsense mutations of the zebrafishhcfc1agene result in contrasting mTor and radial glial phenotypes
Authors: Castro Victoria L. , Paz David , Virrueta Valeria , Estevao Igor L. , Grajeda Brian I. , Ellis Cameron C. , Quintana Anita M. 
Published: October 2022

AbstractMutations in the HCFC1 transcriptional co-factor protein are the cause ofcblXsyndrome and X-linked intellectual disability (XLID).cblXis the more severe disorder associated with intractable epilepsy, abnormal cobalamin metabolism, facial dysmorphia, cortical gyral malformations, and intellectual disability.In vitro,Hcfc1regulates neural precursor (NPCs) proliferation and number, which has been validated in zebrafish. However, conditional deletion ofHcfc1in Nkx2.1+ NPCs increased cell death, reducedGfapexpression, and reduced numbers of GABAergic neurons. Thus, the role of HCFC1 in brain development is not completely understood. Recently, knock-in of both acblX(HCFC1) andcblX-like (THAP11) allele were created in mice. Knock-in of thecblX-like allele was associated with increased expression of proteins required for ribosome biogenesis. However, the brain phenotypes were not comprehensively studied due to sub-viability and therefore, a mechanism underlying increased ribosome biogenesis was not described. We used a missense, a nonsense, and two conditional zebrafish alleles to further elucidate this mechanism during brain development. We observed contrasting phenotypes at the level of Akt/mTor activation, the number of radial glial cells, and the expression of two downstream target genes of HCFC1,asxl1andywhab. Despite these divergent phenotypes, each allele studied demonstrates with a high degree of face validity when compared to the phenotypes reported in the literature. Collectively, these data suggest that individual mutations in the HCFC1 protein result in differential mTor activity which is associated with contrasting cellular phenotypes.
CLOCK and TIMELESS regulate rhythmic occupancy of the BRAHMA chromatin-remodeling protein at clock gene promoters
Authors: Tabuloc Christine A. , Kwok Rosanna S. , Chan Elizabeth C. , Hidalgo Sergio , Cai Yao D. , Chiu Joanna C. 
Published: October 2022

AbstractCircadian clock and chromatin remodeling complexes are tightly intertwined systems that regulate rhythmic gene expression. The circadian clock promotes rhythmic expression, timely recruitment, and/or activation of chromatin remodelers, while chromatin remodelers regulate accessibility of clock transcription factors to the DNA to influence expression of clock genes. We previously reported that the BRAHMA (BRM) chromatin remodeling complex promotes the repression of circadian gene expression inDrosophila.In this study, we investigated the mechanisms by which the circadian clock feeds back to modulate daily BRM activity. Using chromatin immunoprecipitation, we observed rhythmic BRM binding to clock gene promoters despite constitutive BRM protein expression, suggesting that factors other than protein abundance are responsible for rhythmic BRM occupancy at clock-controlled loci. Since we previously reported that BRM interacts with two key clock proteins, CLOCK (CLK) and TIMELESS (TIM), we examined their effect on BRM occupancy to theperiod (per)promoter. We observed reduced BRM binding to the DNA inclknull flies, suggesting that CLK is involved in enhancing BRM occupancy to initiate transcriptional repression at the conclusion of the activation phase. Additionally, we observed reduced BRM binding to theperpromoter in flies overexpressing TIM, suggesting that TIM promotes BRM removal from DNA. This conclusion is further supported by elevated BRM binding to theperpromoter in flies subjected to constant light. In summary, this study provides new insights into the reciprocal regulation between the circadian clock and the BRM chromatin remodeling complex.Author SummaryCircadian clocks are endogenous time-keeping mechanisms that allow organisms to anticipate and adapt to daily changes in their external environment. These clocks are driven by a molecular oscillator that generates rhythms in the expression of many genes, termed clock-controlled genes. The genomic DNA containing these clock-controlled genes are also modified in a rhythmic manner throughout the day. DNA are more tightly packaged with histone proteins when transcription of clock-controlled genes is repressed while the interaction between DNA and histone proteins are more relaxed during transcriptional activation. We found that two key clock proteins, CLOCK and TIMELESS, regulate daily rhythmicity in the binding of BRAHMA, a chromatin remodeler, to DNA spanning clock-controlled genes to facilitate their rhythmic gene expression cycles. Moreover, because TIMELESS is sensitive to light, our study provides new insights into how light can affect DNA structure and gene expression.
Phosphatidylglycerol is the lipid donor for synthesis of phospholipid-linked enterobacterial common antigen
Authors: Morris Kinsey N. , Mitchell Angela M. 
Published: October 2022

ABSTRACTThe gram-negative outer membrane (OM) is an asymmetric bilayer with phospholipids in its inner leaflet and mainly lipopolysaccharide (LPS) in its outer leaflet and is largely impermeable to many antibiotics. InEnterobacterales(e.g.,Escherichia, Salmonella, Klebsiella, Yersinia), the outer leaflet of the OM also contains phosphoglyceride-linked enterobacterial common antigen (ECAPG). This molecule consists of the conserved ECA carbohydrate linked to diacylglycerol-phosphate (DAG-P) through a phosphodiester bond. ECAPGcontributes to the OM permeability barrier and modeling suggests that it may alter the packing of LPS molecules in the OM. Here, we investigate, inEscherichia coliK-12, the reaction synthesizing ECAPGfrom ECA precursor linked to an isoprenoid carrier to identify the lipid donor that provides the DAG-P moiety to ECAPG. Through overexpression of phospholipid biosynthesis genes, we observed alterations expected to increase levels of phosphatidylglycerol (PG) increased synthesis of ECAPG, whereas alterations expected to decrease levels of PG decreased synthesis of ECAPG. We discovered depletion of PG levels in strains that could synthesize ECAPG, but not other forms of ECA, causes additional growth defects, likely due to the buildup of ECA precursor on the isoprenoid carrier inhibiting peptidoglycan biosynthesis. Our results demonstrate ECAPG can be synthesized in the absence of the other major phospholipids (phosphatidylethanolamine and cardiolipin). Overall, these results conclusively demonstrate PG is the lipid donor for the synthesis of ECAPGand provide a key insight into the reaction producing ECAPG. In addition, these results provide an interesting parallel to lipoprotein acylation, which also uses PG as its DAG donor.IMPORTANCEThe outer membrane of gram-negative bacteria is a permeability barrier that prevents the entry of many antibiotics into the cell. However, the pathways responsible for outer membrane biogenesis are potential targets for small molecule development. Here, we investigate the synthesis of a form of enterobacterial common antigen (ECA), ECAPG, found in the outer membrane ofEnterobacteralessuch asEscherichia, Salmonella, Klebsiella, andYersinia. ECAPGconsists of the conserved ECA carbohydrate unit linked to diacylglycerol-phosphate—ECA is the headgroup of a phospholipid. The details of the reaction forming this molecule from ECA linked to an isoprenoid carrier are unknown. We determined that the lipid donor that provides the phospholipid moiety to ECAPGis phosphatidylglycerol. Understanding the synthesis of outer membrane constituents such as ECAPGprovides the opportunity for the development of molecules to increase outer membrane permeability, expanding the antibiotics available to treat gram-negative infections.
Fusion Detection in Microsecretory Adenocarcinoma and Mucoepidermoid Carcinoma Using Chromogenic RNA In Situ Hybridization: A Promising Alternative to DNA-Based Fluorescence In-Situ Hybridization
Authors: Palsgrove Doreen N. , Hosler Calvin , Rooper Lisa M. , Weston Dequan , Day Andrew , Bishop Justin A. , Wang Richard C. 
Published: October 2022

ABSTRACTBackgroundRecent advances in molecular genetics have dramatically improved our understanding of the pathophysiology and classification of salivary gland tumors. The identification of recurrent oncogenic fusions has been especially helpful in distinguishing entities with overlapping histomorphology.MethodsChromogenic RNA in situ hybridization (RNA-ISH) using BaseScope™ technology was performed to detect gene fusions associated with microsecretory adenocarcinoma (MSA),MEF2C::SS18, and mucoepidermoid carcinoma (MEC),CRTC1::MAML2, using probes specific to the exon junctions of theMEF2C::SS18(exon 7 ofMEF2Cto exon 4 ofSS18) andCRTC1::MAML2(exon 1 ofCRTC1to exon 2 ofMAML2) fusion transcripts. Sixteen cases ofMEF2C::SS18fusion-positive MSA, six cases ofCRTC1::MAML2fusion-positive MEC, three cases of fusion-unknown MEC, and one case of fusion-negative MEC were included in the test cohort. Positive signal strength was assessed using a semi-quantitative scoring method as per manufacturer guidelines.ResultsFusion transcripts were detected by RNA-ISH results in 14/16 cases (88%) of fusion-positive MSAs and 3/6 cases (50%) of fusion-positive MEC. Interestingly, 2 cases (67%) of fusion-unknown MEC were also positive by RNA-ISH forCRTC1::MAML2while the fusion-negative MEC was also negative by RNA-ISH. Positivity ranged between 1+ (one dot per cell in ≥5% of tumor cells in one 40X field) and 2+ (two to three dots per cell in ≥5% of tumor cells in one 40X field).ConclusionHere, we provide the first assessment of chromogenic RNA-ISH to detect gene fusions associated with microsecretory adenocarcinoma,MEF2C::SS18, and mucoepidermoid carcinoma,CRTC1::MAML2. Our results highlight the potential for ultrasensitive RNA-ISH to be used as an alternative method of fusion detection for salivary gland malignancies with highly conserved fusion transcript exon junctions. While additional studies are needed to validate the clinical utility of the assay and to determine optimal testing conditions, RNA-ISH may provide a means for restricted fusion analysis in cases with limited material and for pathologists without easy access to conventional molecular diagnostic testing.
Improving classification of correct and incorrect protein-protein docking models by augmenting the training set
Authors: Barradas-Bautista Didier , Almajed Ali , Oliva Romina , Cavallo Luigi , Kalnis Panos 
Published: October 2022

AbstractProtein-protein interactions drive many relevant biological events, such as infection, replication, and recognition. To control or engineer such events, we need to access the molecular details of the interaction provided by experimental 3D structures. However, such experiments take time and are expensive; moreover, the current technology cannot keep up with the high discovery rate of new interactions. Computational modeling, like protein-protein docking, can help to fill this gap by generating docking poses. Protein-protein docking generally consists of two parts, sampling and scoring. The sampling is an exhaustive search of the tridimensional space. The caveat of the sampling produces a large number of incorrect poses, producing a highly unbalanced dataset. This limits the utility of the data to train machine learning classifiers. Using weak supervision, we developed a data augmentation method that we named hAIkal. Using hAIkal, we increased the labeled training data to train several algorithms. We trained and obtained different classifiers; the best classifier has 81% accuracy and 0.51 MCC on the test set, surpassing the state-of-the-art scoring functions.
A peptide strategy for inhibiting different protein aggregation pathways in disease
Authors: Garfagnini Tommaso , Ferrari Luca , Koopman Margreet B. , Halters Sem , Van Kappel Eline , Mayer Guy , Maurice Madelon M. , Rüdiger Stefan G. D. , Friedler Assaf 
Published: October 2022

AbstractProtein aggregation correlates with many human diseases. Protein aggregates differ in shape, ranging from amorphous aggregates to amyloid fibrils. Possibly for such heterogeneity, strategies to develop effective aggregation inhibitors that reach the clinic failed so far. Here, we present a new strategy by which we developed a family of peptides targeting early aggregation stages for both amorphous and fibrillar aggregates of proteins unrelated in sequence and structure. Thus, they act on dynamic precursors before a mechanistic differentiation takes place. Using a peptide array approach, we first identified peptides inhibiting the predominantly amorphous aggregation of a molten globular, aggregation-prone protein, a thermolabile mutant of the Axin tumor suppressor. A series of optimization steps revealed that the peptides activity did not depend on their sequences but rather on their molecular determinants. The key properties that made a peptide active were a composition of 20-30% flexible, 30-40% aliphatic and 20-30% aromatic residues, a hydrophobicity/hydrophilicity ratio close to 1 and an even distribution of residues of different nature throughout the sequence. Remarkably, the optimized peptides also suppressed fibrillation of Tau, a disordered protein that forms amyloids in Alzheimer’s disease, and entirely unrelated to Axin. Our compounds thus target early aggregation stages, independent of the aggregation mechanism, inhibiting both amorphous and amyloid aggregation. Such cross-mechanistic, multi-targeting aggregation inhibitors may be attractive lead compounds against multiple protein aggregation diseases.
Functional Decomposition of Metabolism allows a system-level quantification of fluxes and protein allocation towards specific metabolic functions
Authors: Mori Matteo , Cheng Chuankai , Taylor Brian , Okano Hiroyuki , Hwa Terence 
Published: October 2022

AbstractQuantifying the contribution of individual molecular components to complex cellular processes is a grand challenge in systems biology. Here we establish a general theoretical framework (Functional Decomposition of Metabolism, FDM) to quantify the contribution of every metabolic reaction to metabolic functions, e.g. the biosynthesis of metabolic building blocks such as amino acids. This allows us to obtain a plethora of results forE. coligrowing in different conditions. A detailed quantification of energetic costs for biosynthesis and biomass growth on glucose shows that ATP generated duringde novobiosynthesis of building blocks almost balances the ATP costs of peptide chain polymerization, the single largest energy expenditure for growing cells. This leaves the bulk of energy generated by fermentation and respiration (consuming 1/3 of the glucose intake) during aerobic growth unaccounted for. FDM also enabled the quantification of protein allocated towards each metabolic function, unveiling linear enzyme-flux relations for biosynthesis. These results led us to derive a function-based coarse-grained model to capture global protein allocation and overflow metabolism, without relying on curated pathway annotation or clustering of gene expression data.