bluuebunny/crossref_metadata_2025 huggingface dataset:
From genome wide SNPs to genomic islands of differentiation: the quest for species diagnostic markers in two scleractinian corals,PocilloporaandPorites
Authors: Deshuraud Romane , Ottaviani Alexandre , Poulain Julie , Leprêtre Marine , Beluche Odette , Mahieu Eric , Lebled Sandrine , Belser Caroline , Rouan Alice , Moulin Clementine , Boissin Emilie , Bourdin Guillaume , Iwankow Guillaume , Romac Sarah , Agostini Sylvain , Banaigs Bernard , Boss Emmanuel , Bowler Chris , Vargas Colomban de , Douville Eric , Flores Michel , Furla Paola , Galand Pierre , Lombard Fabien , Pesant Stéphane , Reynaud Stéphanie , Sullivan Matthew B , Sunagawa Shinichi , Thomas Olivier , Troublé Romain , Thurber Rebecca Vega , Voolstra Christian R. , Wincker Patrick , Zoccola Didier , Planes Serge , Allemand Denis , Gilson Eric , Forcioli Didier
Published: October 2022

Coral reefs are of paramount importance in marine ecosystems, where they provide support for a large part of the biodiversity. Being quite sensitive to global changes, they are therefore the prime targets for biodiversity conservation policies. However, such conservation goals require accurate species identification, which are notoriously difficult to get in these highly morphologically variable organisms, rich in cryptic species. There is an acute need for easy-to-use and resolutive species diagnostic molecular markers. The present study builds on the huge sequencing effort developed during the TARA Pacific expedition to develop a genotyping strategy to assign coral samples to the correct species within two coral genera (PoritesandPocillopora). For this purpose, we developed a technique that we called “Divergent Fragment” based on the sequencing of a less than 2kb long diagnostic genomic fragment determined from the metagenomic data of a subset of the corals collected. This method has proven to be rapid, resolvable and cost-effective. Sequencing of PCR fragments nested along the species diagnostic fragment allowed us to assign 232 individuals of the genusPocilloporaand 247 individuals of the genusPoritesto previously identified independent genetic lineages (i.e. species). This genotyping method will allow to fully analyze the coral samples collected across the Pacific during the Tara Pacific expedition and opens technological perspectives in the field of population genomics-guided conservation.
The cellular Notch1 Protein Promotes KSHV reactivation in an Rta-dependent manner
Authors: DeCotiis-Mauro Jennifer , Han Sun M. , Mello Helena , Goyeneche Corey , Marchesini-Tovar Giuseppina , Jin Lianhua , Bellofatto Vivian , Lukac David M.
Published: October 2022

The cellular Notch signal transduction pathway is intimately associated with infections by Kaposi’s sarcoma-associated herpesvirus (KSHV) and other gamma-herpesviruses. RBP-Jk, the cellular DNA binding component of the canonical Notch pathway, is the key Notch downstream effector protein in virus-infected and uninfected animal cells. Reactivation of KSHV from latency requires the viral lytic switch protein, Rta, to form complexes with RBP-Jk on numerous sites within the viral DNA. Constitutive Notch activity is essential for KSHV pathophysiology in models of Kaposi’s sarcoma (KS) and Primary Effusion Lymphoma (PEL), and we demonstrate that Notch1 is also constitutively active in infected Vero cells. Although the KSHV genome contains >100 RBP-Jk DNA motifs, we show that none of the four isoforms of activated Notch can productively reactivate the virus from latency in a highly quantitative trans-complementing reporter virus system. Nevertheless, Notch contributed positively to reactivation because broad inhibition of Notch1-4 with gamma secretase inhibitor (GSI) or expression of dominant negative mastermind-like1 (dnMAML1) coactivators severely reduced production of infectious KSHV from Vero cells. Reduction of KSHV production is associated with gene specific reduction of viral transcription in both Vero and PEL cells. Specific inhibition of Notch1 by siRNA partially reduces production of infectious KSHV, and NICD1 forms promoter-specific complexes with viral DNA during reactivation. We conclude that constitutive Notch activity is required for robust production of infectious KSHV, and our results implicate activated Notch1 as a pro-viral member of a MAML1/RBP-Jk/DNA complex during viral reactivation.

Importance

Kaposi’s sarcoma-associated herpesvirus (KSHV) manipulates the host cell oncogenic Notch signaling pathway for viral reactivation from latency and cell pathogenesis. KSHV reactivation requires that the viral protein Rta functionally interacts with RBP-Jk, the DNA binding component of the Notch pathway, and with promoter DNA to drive transcription of productive cycle genes. We show that the Notch pathway is constitutively active during KSHV reactivation and is essential for robust production of infectious virus progeny. Inhibiting Notch during reactivation reduces expression of specific viral genes yet does not affect growth of the host cells. Although Notch cannot reactivate KSHV alone, the requisite expression of Rta reveals a previously unappreciated role for Notch in reactivation. We propose that activated Notch cooperates with Rta in a promoter-specific manner that is partially programmed by Rta’s ability to redistribute RBP-Jk DNA binding to the virus during reactivation.
GSearch: Ultra-Fast and Scalable Microbial Genome Search by Combining K-mer Hashing with Hierarchical Navigable Small World Graphs
Authors: Zhao Jianshu , Both Jean Pierre , Rodriguez-R Luis M. , Konstantinidis Konstantinos T.
Published: October 2022

Genome search and/or classification is a key step in microbiome studies and has recently become more challenging due to the increasing number of available (reference) genomes and the fact that traditional methods do not scale well with large databases. By combining k-mer hashing-based probabilistic data structures (e.g., (Prob/Super/Densified)-MinHash or SetSketch) to estimate genomic distance, with a graph-based nearest neighbor search algorithm (called Hierarchical Navigable Small World Graphs, or HNSW), we created a new data structure and developed an associated computer program, GSearch, that is orders of magnitude faster than alternative tools while maintaining high accuracy and low memory usage. For example, GSearch can identify/classify 8,000 query genomes against all available microbial or viral genomes (n=∼318,000 or ∼3,000,000) within a few minutes on a personal laptop, using only ∼6GB of memory or less (e.g., 2.5G via SetSketch). Notably, GSearch will be even faster compared to other tools with even larger database size due to O(log(N)) time complexity and will scale well with billions of database genomes based on a database splitting strategy. Further, GSearch implements a three-step classification pipeline that accounts for the degree of novelty of query genomes relative to the database genome to maximize specificity and sensitivity. Therefore, GSearch solves a major bottleneck of microbiome studies that require genome search and/or classification of microbial or viral genomes. GSearch is available at:https://github.com/jean-pierreBoth/gsearch
Transcriptomics reveals amygdala neuron regulation by fasting and ghrelin thereby promoting feeding
Authors: Peters Christian , He Songwei , Fermani Federica , Lim Hansol , Mayer Christian , Klein Rüdiger
Published: October 2022

The central amygdala (CeA) consists of numerous genetically-defined inhibitory neurons that control defensive and appetitive behaviors including feeding. Transcriptomic signatures of cell types and their links to function remain poorly understood. Using single-nucleus RNA-sequencing, we describe nine CeA cell clusters, of which four are mostly associated with appetitive and two with aversive behaviors. To analyze the activation mechanism of appetitive CeA neurons, we characterized Serotonin receptor 2a (Htr2a)-expressing neurons (CeAHtr2a) that comprise three appetitive clusters and were previously shown to promote feeding. In vivo calcium imaging revealed that CeAHtr2aneurons are activated by fasting, the hormone ghrelin, and the presence of food. Moreover, these neurons are required for the orexigenic effects of ghrelin. Appetitive CeA neurons responsive to fasting and ghrelin project to the parabrachial nucleus (PBN) causing inhibition of target PBN neurons. These results illustrate how the transcriptomic diversification of CeA neurons relates to fasting and hormone-regulated feeding behavior.

Highlights

Transcriptomics reveals four appetitive, two aversive and three unknown CeA clusters

Ghrelin and fasting activate CeAHtr2aneurons comprising three appetitive clusters

Ghrelin’s orexigenic effects require the activity of appetitive CeA neurons

CeA→PBN inhibition is necessary for ghrelin/fasting induced feeding
Blood and site of disease inflammatory profiles differ in HIV-1-infected pericardial tuberculosis patients
Authors: Mutavhatsindi Hygon , Du Bruyn Elsa , Ruzive Sheena , Howlett Patrick , Sher Alan , Mayer-Barber Katrin D. , Barber Daniel L. , Ntsekhe Mpiko , Wilkinson Robert J. , Riou Catherine
Published: October 2022

Objectives

To better understand the pathogenesis of pericardial tuberculosis (PCTB), we sought to characterize the systemic inflammatory profile in HIV-1-infected participants with latent TB infection (LTBI), pulmonary TB (PTB) and PCTB.

Methods

Using Luminex, we measured 39 analytes in pericardial fluid (PCF) and paired plasma from 18 PCTB participants, and plasma from 16 LTBI and 20 PTB. Follow-up plasma samples were also obtained from PTB and PCTB participants. HLA-DR expression on Mtb-specific CD4 T cells was measured in baseline samples using flow cytometry.

Results

Assessment of the overall systemic inflammatory profile by principal component analysis showed that the inflammatory profile of active TB participants was distinct from the LTBI group, while PTB patients could not be distinguished from those with PCTB. In the LTBI group, 12 analytes showed a positive association with plasma HIV-1 viral load, and most of these associations were lost in the diseased groups. When comparing the inflammatory profile between PCF and paired blood, we found that the concentrations of most analytes (24/39) were elevated at site of disease. However, the inflammatory profile in PCF partially mirrored inflammatory events in the blood. After TB treatment completion, the overall plasma inflammatory profile reverted to those observed in the LTBI group. Lastly, HLA-DR expression showed the best performance for TB diagnosis compared to previously described biosignatures built from soluble markers.

Conclusion

Our results describe the inflammatory profile associated with PTB and PCTB and emphasize the potential role of HLA-DR as a promising biomarker for TB diagnosis.
Loss of altruism in the social amoebaDictyostelium discoideumis associated with the G protein-coupled receptorgrlG
Authors: Walker Laura M. , Sherpa Rintsen N. , Ivaturi Sindhuri , Brock Debra A. , Walker Jason R. , Strassmann Joan E. , Queller David C.
Published: October 2022

Aggregative multicellularity relies on cooperation among individual cells to form a multicellular body. InDictyostelium discoideumthis cooperation is maintained by high relatedness. Previous work showed that experimental evolution under low-relatedness resulted in an increase of cheaters (cells that contribute proportionally more to spores than to the sterile stalk) and that many clones completely lost cooperation and the ability to form fruiting bodies. Here, we investigate the genomic changes underlying the evolution of the cheating phenotype using whole-genome sequencing and variant analysis of these previously evolvedD. discoideumlines. We identified 38 single nucleotide polymorphisms in 29 genes, none of which have been previously implicated in cheating. Each gene has one variant except for the G protein-coupled receptorgrlG, which has at least one variant in over half of the lines. Upon identifying the parallel evolution ofgrlG, we screened additional clones to investigate the correlation between variants in the gene and the loss of cooperation (identified by the inability to form a fruiting body). We found that variants in the 5’ half ofgrlGthat impact the signal peptide or extracellular binding domain are significantly associated with the loss of cooperation (non-fruiting); the association was not significant in the 3’ half of the gene. This suggests that the loss ofgrlGwas adaptive under low-relatedness and that the 5’ half of the gene in particular is important for cooperation and multicellular development. This confirms the importance of high relatedness in the evolution of altruism in the social amoebaD. discoideum.
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

Mutation 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

Cephalopod 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

Flying 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

Risk 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

Mutations 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

Circadian 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 Summary

Circadian 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

The 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.

IMPORTANCE

The 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

Background

Recent 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.

Methods

Chromogenic 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.

Results

Fusion 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).

Conclusion

Here, 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

Protein-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

Protein 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

Quantifying 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.
Distinct tumor-immune ecologies in NSCLC patients predict progression and define a clinical biomarker of therapy response
Authors: Prabhakaran Sandhya , Gatenbee Chandler , Robertson-Tessi Mark , Beg Amer A. , Gray Jhanelle , Antonia Scott , Gatenby Robert A. , Anderson Alexander R. A.
Published: October 2022

1.

Lung cancer is the leading cause of cancer death worldwide with 1.76 million people die as a result of the disease yearly. Analyzing high-throughput multiplexed images of tissues has recently emerged as a routine clinical procedure for early detection, clinical cancer diagnosis, treatment planning and prognosis for many cancer types including non-small cell lung cancer (NSCLC). Multiplexed images enable the precise interpretation of spatial distribution of cells and cellular states and the characterization of tumor-immune interactions in situ and at the single-cell level. Through two cornerstone image processing techniques - cell segmentation and image tiling generating quadrats - we perform an in-depth analysis of multiplexed images based on the frequency, phenotype, and spatial distribution of immune and tumor cells within the immune landscape of NSCLC. Multiplexed images were obtained from nine patients with advanced/metastatic NSCLC with progression who were treated with an oral HDAC inhibitor (vorinostat) combined with a PD-1 inhibitor (pembrolizumab). Images were collected from all patients both pre- and on-treatment (days 15-21). We profile ∼100K cells and ∼10K quadrats from tumor and adjacent tissues to characterize cellular composition and spatial neighborhoods and elucidate that distinct spatial cellular ecologies exist across progressive disease (PD) and stable disease (SD) patients where tumors of PD patients are characterized by a highly suppressed immune environment prior to treatment enabling higher chances of disease progression during treatment. These fundamentally distinct architectures across PD and SD patients enable disease progression prediction and clinical biomarker identification. Our results also provide a potential companion biomarker for PD-L1 in NSCLC. Validating the biomarker and its feasibility will require further in-depth investigation.
Unsupervised Discovery of Ancestry Informative Markers and Genetic Admixture Proportions in Biobank-Scale Data Sets
Authors: Ko Seyoon , Chu Benjamin B. , Peterson Daniel , Okenwa Chidera , Papp Jeanette C. , Alexander David H. , Sobel Eric M. , Zhou Hua , Lange Kenneth L.
Published: October 2022

Admixture estimation plays a crucial role in ancestry inference and genomewide association studies (GWAS). Computer programs such as ADMIXTURE and STRUCTURE are commonly employed to estimate the admixture proportions of sample individuals. However, these programs can be overwhelmed by the computational burdens imposed by the 105to 106samples and millions of markers commonly found in modern biobanks. An attractive strategy is to run these programs on a set of ancestry informative SNP markers (AIMs) that exhibit substantially different frequencies across populations. Unfortunately, existing methods for identifying AIMs require knowing ancestry labels for a subset of the sample. This supervised learning approach creates a chicken and the egg scenario. In this paper, we present an unsupervised, scalable framework that seamlessly carries out AIM selection and likelihood-based estimation of admixture proportions. Our simulated and real data examples show that this approach is scalable to modern biobank data sets. Our implementation of the method is called OpenADMIXTURE.
Genomic Variation, Population Structure, and Gene Flow across Asian Pikas
Authors: Dahal Nishma , Romine Melia G , Khatiwada Sunita , Ramakrishnan Uma , Lamichhaney Sangeet
Published: October 2022

Asian pikas have one of the most complex systematics and evolutionary history. The Himalayas is an important habitat for Asian pikas as it hosts 23 – 25% of pika’s global diversity and has provided the ancestral training ground for local adaptation to high altitudes. They are one of the most abundant species in the Himalayas and Qinghai Tibetan Plateau (QTP), however genetic studies to explore their population structure and evolution are limited. Here, we utilize a population genomics approach using ~28,000 genome-wide SNP markers obtained from restriction-site associated DNA sequencing in six species of Asian Pikas(Ochotonaspp.)that are distributed across the Himalayas and neighboring high-altitude mountains. We examined intra- and inter-species genetic diversity, population structure, phylogenetic history and explored processes that shaped the current genetic diversity of Pikas across the Himalayas. We identified low nucleotide diversity and high inbreeding coefficient across all species which possibly indicated decreasing population size in these species. We also identified extensive evidence of gene flow (both historic and contemporary) across these species. Our findings indicate that inter-species gene flow is a key evolutionary process that has been countering the negative effect of low genetic diversity among Asian pikas.
The conformational ensemble of an intrinsically disordered protein explains peak shapes under DNP conditions
Authors: Kragelj Jaka , Dumarieh Rania , Xiao Yiling , Frederick Kendra K.
Published: October 2022

Elucidating the conformational preferences of regions of intrinsic disorder in biologically relevant contexts represents a frontier of structural biology. The sensitivity enhancements conferred by DNP enable structural studies of proteins in native contexts by MAS NMR. However, DNP requires low temperatures which results in broad peaks, particularly for for regions of intrinsic disorder. We describe an approach to predict and interpret peak shapes for frozen regions of intrinsic disorder in terms of dihedral angle populations. We demonstrate the method using the protein a-synuclein. This approach can be used to obtain experimental structural restraints for regions of intrinsic disorder in both simplified and biological settings, providing information that eludes characterization by diffraction-based methods as well as solution-state NMR spectroscopy and molecular dynamics due to molecular size limitations.
Transcription-replication interactions reveal principles of bacterial genome regulation
Authors: Pountain Andrew W. , Jiang Peien , Yao Tianyou , Homaee Ehsan , Guan Yichao , Podkowik Magdalena , Shopsin Bo , Torres Victor J. , Golding Ido , Yanai Itai
Published: October 2022

Organisms determine the transcription rates of thousands of genes through a few modes of regulation that recur across the genome. These modes interact with a changing cellular environment to yield highly dynamic expression patterns. In bacteria, the relationship between a gene’s regulatory architecture and its expression is well understood for individual model gene circuits. However, a broader perspective of these dynamics at the genome-scale is lacking, in part because bacterial transcriptomics have hitherto captured only a static snapshot of expression averaged across millions of cells. As a result, the full diversity of gene expression dynamics and their relation to regulatory architecture remains unknown. Here we present a novel genome-wide classification of regulatory modes based on each gene’s transcriptional response to its own replication, which we term the Transcription-Replication Interaction Profile (TRIP). We found that the response to the universal perturbation of chromosomal replication integrates biological regulatory factors with biophysical molecular events on the chromosome to reveal a gene’s local regulatory context. While the TRIPs of many genes conform to a gene dosage-dependent pattern, others diverge in distinct ways, including altered timing or amplitude of expression, and this is shaped by factors such as intra-operon position, repression state, or presence on mobile genetic elements. Our transcriptome analysis also simultaneously captures global properties, such as the rates of replication and transcription, as well as the nestedness of replication patterns. This work challenges previous notions of the drivers of expression heterogeneity within a population of cells, and unearths a previously unseen world of gene transcription dynamics.
Forest bird decline and community change over 19 years in long-isolated South Asian tropical rainforest fragments
Authors: Surendra Akshay , Raman T. R. Shankar
Published: October 2022

Recent evidence of forest bird declines worldwide is attributed to climate change and its interactive effects with recent land-use changes such as forest loss and fragmentation, and avian life-history traits. In Asian tropical forests, such effects are poorly understood as long-term data are lacking from fragments that are long-isolated rather than recently fragmented. Here, we use data from ~2000 point-counts from bird surveys carried out between 2000 – 2005 and 2019 in 19 long-isolated (~80 y) South Asian tropical rainforest fragments to examine changes in bird species richness, density, and composition in relation to fragment area (0.7 – 4310 ha), habitat structure, and time. Over the 19 y timespan, despite stable fragment areas, we uncovered a 29% decline in rainforest bird density and 7% decline in individual-rarefied species richness of rainforest birds, while density and richness of open-country birds remained stable. With increasing fragment area, rainforest bird species richness (jackknife estimate) increased, while open country bird richness (individual-rarefied) and density decreased. Larger fragments housed more compositionally stable bird communities, while poorer habitat was associated with lower diversity of rainforest birds but higher diversity, density, and compositional variation of open-country birds. Threshold analysis however indicated relatively small area thresholds (~20 ha) for rainforest bird species abundance. Besides identifying alarming declines in rainforest birds, the study confirms some but not all predictions for bird diversity in long-isolated forest fragments with stable forest-matrix boundaries, indicating that small fragments and habitat quality also matter.
Methods for Evaluating Effects of Transgenes for Quantitative Traits
Authors: Linares Julien F. , Coles Nathan D. , Mo Hua , Habben Jeffrey E. , Humbert Sabrina , Messina Carlos , Tang Tom , Cooper Mark , Gho Carla , Carrasco Ricardo , Carter Javier , Wicher Flounders Jillian , Brummer E. Charles
Published: October 2022

Transgenes that improve quantitative traits have traditionally been evaluated in one or a few genetic backgrounds across multiple environments. However, testing across multiple genetic backgrounds can be equally important to accurately quantify the value of a transgene for breeding objectives. Creating near-isogenic lines across a wide germplasm space is costly and time consuming, which renders it impractical during early stages of testing. In this experiment, we evaluate three approaches to sample the genetic space while concurrently testing across environments. We created both transgenic and non-transgenic doubled haploid lines, F2:3lines, and bulk F3families to determine if all methods resulted in similar estimation of transgene value and to identify the number of yield trial plots from each method necessary to obtain a stable estimate of the transgene value. With one exception, the three methods consistently estimated a similar effect of the transgene. We suggest that bulked F3lines topcrossed to a tester inbred is the most effective method to estimate the value of a transgene across both genetic space and environments.