title: The replication stress response suppresses mutation rates in mismatch repair deficient budding yeast and human cancers

abstract: Elevated mutation rate is a hallmark of mismatch repair (MMR) deficient cells and tumours. This includes microsatellite instability (MSI), which is caused by insertions and deletions in mono-and dinucleotide repeats. MSI rates, however, are highly variable across MMR-deficient tumours. Here we show that mutation rates are genetically regulated in MMR-deficient cells. A genome-wide deletion screen in budding yeast revealed that 3% gene deletions caused mutation rates to be further elevated, whereas 11% reduced mutation rates. The genes causing an elevation are enriched for DNA repair and replication processes, whereas deletion of genes implicated in transcriptional processes reduce mutation rates. A pan-cancer analysis of MSI revealed that mutations in replication stress response (ATR, TOPBP1, CHEK1) or DNA repair genes (RAD50, TOP3A) was associated with extreme rates of MSI in MMR-defective tumours, but not when mutated on their own. Since replication stress, DNA damage and repair activities are cell-type specific, this may account for highly variable mutation rates associated with different MMR-deficient tumours.


title: Rewiring of liver diurnal transcriptome rhythms by triiodothyronine (T3) supplementation

abstract: Diurnal (i.e., 24-hour) physiological rhythms depend on transcriptional programs controlled by a set of circadian clock genes/proteins. Systemic factors like humoral and neuronal signals, oscillations in body temperature, and food intake align physiological circadian rhythms with external time. Thyroid hormones (THs) are major regulators of circadian clock target processes such as energy metabolism, but little is known about how fluctuations in TH levels affect the circadian coordination of tissue physiology. In this study, a high triiodothyronine (T3) state was induced in mice by supplementing T3 in the drinking water, which affected body temperature, and oxygen consumption in a time-of-day dependent manner. 24-hour transcriptome profiling of liver tissue identified 37 robustly and time independently T3 associated transcripts as potential TH state markers in the liver. Such genes participated in xenobiotic transport, lipid and xenobiotic metabolism. We also identified 10 - 15 % of the liver transcriptome as rhythmic in control and T3 groups, but only 4 % of the liver transcriptome (1,033 genes) were rhythmic across both conditions - amongst these several core clock genes. In-depth rhythm analyses showed that most changes in transcript rhythms were related to mesor (50%), followed by amplitude (10%), and phase (10%). Gene set enrichment analysis revealed TH state dependent reorganization of metabolic processes such as lipid and glucose metabolism. At high T3 levels, we observed weakening or loss of rhythmicity for transcripts associated with glucose and fatty acid metabolism, suggesting increased hepatic energy turnover. In sum, we provide evidence that tonic changes in T3 levels restructure the diurnal liver metabolic transcriptome independent of local molecular circadian clocks.

category: physiology


title: PILS proteins provide a homeostatic feedback on auxin signaling output

abstract: Auxin is a crucial regulator of plant growth and development. Multiple internal and external signals converge at the regulation of auxin metabolism, intercellular transport, and signaling (Pernisova and Vernoux, 2021; Anfang and Shani, 2021). Considering this complexity, it remains largely unknown how plant cells monitor and ensure the homeostasis of auxin responses. PIN-LIKES (PILS) intracellular auxin transport facilitators at the endoplasmic reticulum (ER) are suitable candidates to buffer cellular auxin responses, because they limit nuclear abundance and signaling of auxin (Barbez et al., 2012; Beziat et al., 2017; Feraru et al., 2019; Sun et al., 2020). We used forward genetics to identify mechanisms that define the PILS6 protein abundance and thereby auxin signaling outputs. We screened for gloomy and shiny pils (gasp) mutants that define the levels of PILS6-GFP under a constitutive promoter. In this study, we show that GASP1 encodes for an uncharacterized RING/U-box superfamily protein and impacts on auxin signaling output. We conclude that the low auxin signaling in gasp1 mutants correlates with reduced abundance of PILS proteins, such as PILS5 and PILS6, which consequently balances auxin-related phenotypes. In agreement, we show that high and low auxin conditions increase and reduce PILS6 protein levels, respectively. Accordingly, non-optimum auxin concentrations are buffered by alterations in PILS6 abundance, consequently leading to homeostatic auxin output regulation. We envision that this feedback mechanism provides robustness to auxin-dependent plant development.

category: plant biology


title: GB_SynP: a modular dCas9-regulated synthetic promoter collection for fine-tuned recombinant gene expression in plants.

abstract: Programable transcriptional factors based on the CRISPR architecture are becoming commonly used in plants for endogenous gene regulation. In plants, a potent CRISPR tool for gene induction is the so-called dCasEV2.1 activation system, which has shown remarkable genome-wide specificity combined with a strong activation capacity. To explore the ability of dCasEV2.1 to act as a transactivator for orthogonal synthetic promoters, a collection of DNA parts was created (GB_SynP) for combinatorial synthetic promoter building. The collection includes (i) minimal promoter parts with the TATA box and 5UTR regions, (ii) proximal parts containing single or multiple copies of the target sequence for the gRNA, thus functioning as regulatory cis boxes, and (iii) sequence-randomized distal parts that ensure the adequate length of the resulting promoter. A total of 35 promoters were assembled using the GB_SynP collection, showing in all cases minimal background and predictable activation levels depending on the proximal parts used. GB_SynP was also employed in a combinatorial expression analysis of an auto-luminescence pathway in Nicotiana benthamiana, showing the value of this tool in extracting important biological information such as the determination of the limiting steps in an enzymatic pathway.

category: synthetic biology


title: The activation of mGluR4 rescues parallel fiber LTP, motor learning and social behavior in a mouse model of Fragile X Syndrome

abstract: Fragile X patients and mice lacking the Fragile X Mental Retardation Protein (FMRP) suffer from multiple behavioral alterations, including deficits in motor learning. We found that enhanced synaptic vesicle (SV) docking in cerebellar parallel fiber to Purkinje cell Fmr1KO synapses was associated with enhanced asynchronous release, which not only occludes further potentiation, but it also compromises presynaptic parallel fiber long-term potentiation (PF-LTP). A reduction in extracellular Ca2+ restored the readily releasable pool (RRP) size, rescuing {beta} adrenergic receptor-mediated potentiation and parallel fiber LTP. Interestingly, VU 0155041, a selective positive allosteric modulator of mGluR4, also restored both the RRP size and parallel fiber LTP. Moreover, when injected into Fmr1KO mice, VU 0155041 improved motor learning in skilled reaching, classical eyeblink conditioning and vestibuloocular reflex (VOR) tests, as well as improving the social behavior of these mice. Thus, pharmacological activation of mGluRs may offer therapeutic relief for motor learning and social deficits in Fragile X Syndrome.

category: neuroscience


title: 5' Untranslated mRNA Regions Allow Bypass of Host Cell Translation Inhibition by Legionella pneumophila

abstract: Legionella pneumophila grows within membrane-bound vacuoles in alveolar macrophages during human disease. Pathogen manipulation of the host cell is driven by bacterial proteins translocated through a type IV secretion system (T4SS). Although host protein synthesis during infection is arrested by the action of several of these translocated effectors, translation of a subset of host proteins predicted to restrict the pathogen is maintained. To identify the spectrum of host proteins selectively synthesized after L. pneumophila challenge, macrophages infected with the pathogen were allowed to incorporate the amino acid analog azidohomoalanine (AHA) during a two-hour time window, and newly synthesized macrophage proteins were isolated by orthogonal chemistry followed by mass spectrometry. Among the proteins isolated were interferon-stimulated genes (ISGs) as well as proteins translated from highly abundant transcripts. Surprisingly, a large number of the identified proteins were from low abundance transcripts. These proteins were predicted to be among the most efficiently translated per unit transcript in the cell based on ribosome profiling datasets. To determine if high ribosome loading was a consequence of efficient translation initiation, the 5 untranslated regions (5UTR) of transcripts having the highest and lowest predicted loading levels were inserted upstream of a reporter, and translation efficiency was determined in response to L. pneumophila challenge. The efficiency of reporter expression largely correlated with predicted ribosome loading and lack of secondary structure. Therefore, determinants in the 5UTR allow selected host cell transcripts to overcome a pathogen-driven translation blockade.

category: microbiology


title: Long Term Virucidal Activity of Rosin Soap on Surfaces

abstract: Microbiocidal products for decontaminating both animate and inanimate surfaces that may act as vectors for disease transmission are a well-established method for inactivating viruses of clinical significance. There are a wide variety of such microbiocidal products that can be purchased commercially, many with different active ingredients. We have recently shown that Rosin soap (derived from Tall Oil that has been produced by trees) is a highly effective virucidal product against enveloped viruses like influenza A virus and SARS-CoV-2 when tested in solution. Here we further demonstrate that Rosin soap retains its virucidal activity against influenza A virus and SARS-CoV-2 when applied to surfaces, such as plastic, glass and steel, either before or after virus inoculation. The virucidal activity extended out to seven days post administration. Together, our results show the potential for Rosin soap-based disinfectants to be used as a surface coating to protect against clinically relevant enveloped viruses, such as influenza viruses and coronaviruses.


title: Partial refuges from biological control due to intraspecific variation in protective host traits: a case study with the egg parasitoid Trissolcus japonicus

abstract: Predicting how much of a host or prey population may be attacked by their natural enemies is fundamental to several subfields of applied ecology, particularly biological control of pest organisms. Hosts or prey can occupy refuges from natural enemy attack, but habitat or ecological refuges are challenging or impossible to predict in a laboratory setting - which is often where efficacy and specificity testing of candidate biological control agents is done. Here we explore how intraspecific variation in continuous traits that confers some protection from natural enemy attack - even after the natural enemy has encountered the prey - could provide partial refuges. The size of these trait-based refuges should depend on the relationship between trait values and host/prey susceptibility to natural enemy attack, and on how common different trait values are within a host/prey population. These can be readily estimated in laboratory testing of natural enemy impact on target or non-target prey or hosts as long as sufficient host material is available. We provide a general framework for how intraspecific variation in protective host traits could be integrated into biological control research, specifically with reference to non-target testing as part of classical biological control programs. As a case study, we exposed different host clutch sizes of target (pest) and non-target (native species) stink bug (Hemiptera: Pentatomidae) species to a well-studied exotic biocontrol agent, the egg parasitoid Trissolcus japonicus (Hymenoptera: Scelionidae). Although we observed several behavioural and reproductive responses to variation in host egg mass size by T. japonicus, they did not translate to increases in predicted refuge size (proportion host survival) large enough to change the conclusions of non-target testing. We encourage researchers to investigate intraspecific variation in a wider variety of protective host and prey traits and their consequences for refuge size.

category: ecology


title: Sorting Single T cells based on Secreted Cytokines and Surface Markers using Hydrogel Nanovials

abstract: Immune cell function is intrinsically linked to secreted factors which enable cells to communicate with neighboring or distant cells to coordinate a response. The ability to secrete cytokines also can help define the population of cells with therapeutic potential in emerging cell therapies, such as chimeric antigen receptor (CAR)-T cell therapies. Polyfunctional cells that can secrete more than one cytokine have been found to play an outsized role in therapeutic efficacy. While there are a variety of techniques to analyze cellular secretions from individual polyfunctional cells, there are no widely-available approaches to sort viable cells based on this phenotype. Here, we apply lab on a particle technology to the analysis and sorting of T cells based on a combination of secreted factors, interferon gamma (IFN-{gamma}), tumor necrosis factor alpha (TNF-) and interleukin 2 (IL-2) and surface markers (CD8+ and CD4+). Cells are selectively loaded into the antibody-functionalized cavity of micro-hydrogel particles, called nanovials, where secreted cytokines are captured and fluorescently stained. By leveraging standard fluorescence activated cell sorters and using fluorescence pulse area/height information we can distinguish between fluorescence signals on the nanovial cavities and on cells, and are able to process greater than 1 million nanovials in one hour of sorting. The frequency of multi-cytokine secreting cells was correlated with surface marker expression, and biased towards CD4+ T cells. CD8+ cells that secreted more than one cytokine, were biased towards IFN-{gamma} and TNF- with fewer CD8+ cells secreting IL-2. The majority of cells with a polyfunctional phenotype that were sorted remained viable and regrew following sorting. This nanovial cytokine secretion assay can be applied to sort antigen-specific T cells or CAR-T cells based on their functional engagement with cognate antigens or peptide-major histocompatibility complexs (MHCs), enabling discovery of functional CARs or T cell receptors and deeper investigation into the molecular underpinnings of single T cell function.

category: bioengineering


title: Genomic analyses identify significant biological processes in DDX21-mediated colorectal cancer cells

abstract: Colorectal cancer is the third most common cancer in the US. There has been an incline in the number of young patients with colorectal cancer due to unclear reasons at this point in time. Currently, DEAD-box RNA helicase protein DDX21 is identified as a prognosis marker for early-stage colorectal cancer. However, the mechanism of DDX21 mediated-colorectal cancer is still unknown. Here, our objective is to determine the key molecules and signaling by analyzing the RNA-seq data. The GSE184726 was created by the Illumina NovaSeq 6000 (Homo sapiens). The KEGG and GO analyses indicated Neuroactive ligand-receptor interaction and Ras signaling pathway were the key signaling pathways during the knockdown of DDX21 in colorectal cancer. Moreover, we identified several interactive genes including PTPRC, FN1, ITGAM, RAD51, TRAF6, CCNB1, FOXP3, CCNA2, HIST2H2AC, and HSPA5. Our study may provide new insights into the treatment of colorectal cancer.

category: bioinformatics


title: Early steps of embryo implantation are regulated by exchange of extracellular vesicles between the embryo and the endometrium

abstract: In early pregnancy, as the embryo arrives in the uterus, intensive communication between the embryo and uterus begins. Hundreds of molecules are known to be involved, but despite numerous findings, full understanding of the complexity of the embryo-maternal dialog remains elusive. Recently, extracellular vesicles, nanoparticles able to transfer functionally active cargo between cells, have emerged as important players in cell-cell communication, and as such, they have gained great attention over the past decade also in reproductive biology. Here we use a domestic animal model (Sus scrofa) with an epitheliochorial, superficial type of placentation because of its advantage in studding uterine luminal fluid extracellular vesicles. We show that during early pregnancy, the uterine lumen is abundant with extracellular vesicles that carry a plethora of miRNAs able to target genes involved in embryonic and organismal development. These extracellular vesicles, upon the delivery to primary trophoblast cells, affect genes governing development as well as cell-to-cell signaling and interactions, consequently having an impact on trophoblast cell proliferation, migration, and invasion. We conclude that exchange of a unique population of extracellular vesicles and their molecular cargo at the maternal-embryo interface is the key to the success of embryo implantation and pregnancy.


title: BMAL1 loss in oligodendroglial lineage cells dysregulates myelination and sleep

abstract:
Myelination depends on maintenance of oligodendrocytes that arise from oligodendrocyte precursor cells (OPCs). We show that the dynamic nature of oligodendroglia and myelination are regulated by the circadian transcription factor BMAL1. Bmal1 knockdown in OPCs during development - but not adulthood - decreases OPC proliferation, whereas BMAL1 regulates OPC morphology throughout life. OPC-specific Bmal1 deficiency impairs remyelination in an age-dependent manner, suggesting that age-associated decrements in circadian regulation of oligodendroglia may contribute to the deficient remyelination potential in demyelinating diseases like multiple sclerosis (MS). This oligodendroglial dysregulation and dysmyelination increase sleep fragmentation in OPC-specific Bmal1 knockout mice, and sleep fragmentation is causally associated with MS. These findings have broad mechanistic and therapeutic implications for numerous brain disorders that include both myelin and sleep phenotypes.

One-Sentence SummaryBMAL1 regulates the homeostatic maintenance of oligodendroglia and myelin, that subsequently controls sleep architecture.


title: Evidence for the presence and diagnostic utility of SPM in human peripheral blood

abstract:
We thank ODonnell et al, for their comments on our contribution and are grateful to be afforded this opportunity to formally respond to their critique24.

We are surprised by the authors assertion relating to the biological relevance of SPM because a simple literature search for related terms such as  resolvin in PubMed yields an abundance (>1,420 publications) of evidence supporting the potent biological activities and the diagnostic potential of some of these mediators. Several co-authors of the ODonnells et al manuscript, have published on the resolvins and SPMs, including some publications within recent weeks. Importantly, ODonnell et al, misreport as well as mis-apply criteria for peak identification reported in the Gomez et al, publication which lead to the flawed analysis they performed.

In this response therefore, we provide a step-by-step clarification of the methodologies used in Gomez et al, and a side-by-side comparison of the underlying data to clarify any confusion. We also demonstrate that using the orthogonal criteria discussed by ODonnell et al, we obtain essentially identical results thus providing additional validation of our techniques and support the conclusions.

category: immunology


title: A guide for membrane potential measurements in Gram-negative bacteria using voltage-sensitive dyes

abstract: Transmembrane potential is one of the main bioenergetic parameters of bacterial cells, and is directly involved in energising key cellular processes such as transport, ATP synthesis, and motility. The most common approach to measure membrane potential levels is through use of voltage-sensitive fluorescent dyes. Such dyes either accumulate or are excluded from the cell in a voltage-dependent manner, which can be followed by means of fluorescence microscopy, flow cytometry, or fluorometry. Since the cells ability to maintain transmembrane potential relies upon low membrane ion conductivity, voltage-sensitive dyes are also highly sensitive reporters for the activity of membrane-targeting antibacterials. However, the presence of an additional membrane layer in Gram-negative (diderm) bacteria significantly complicates their use. In this manuscript, we provide guidance on how membrane potential and its changes can be reliably monitored in Gram-negatives using the voltage-sensitive dye DiSC3(5). We also discuss the confounding effects caused by the presence of the outer membrane, or by measurements performed in buffers rather than growth medium. We hope that the discussed methods and protocols provide an easily accessible basis for the use of voltage-sensitive dyes in Gram-negative organisms, and raise awareness of potential experimental pitfalls associated with their use.


title: Characterization of the DNA binding domain of StbA, a key protein of a new type of DNA segregation system

abstract: Low-copy-number plasmids require sophisticated genetic devices to achieve efficient segregation of plasmid copies during cell division. Plasmid R388 uses a unique segregation mechanism, based on StbA, a small multifunctional protein. StbA is the key protein in a segregation system not involving a plasmid-encoded NTPase partner, it regulates the expression of several plasmid operons, and it is the main regulator of plasmid conjugation. The mechanisms by which StbA, together with the centromere-like sequence stbS, achieves segregation, is largely uncharacterized. To better understand the molecular basis of R388 segregation, we determined the crystal structure of the conserved N-terminal domain of StbA to 1.9 [A] resolution. It folds into an HTH DNA-binding motif, structurally related to that of the PadR subfamily II of transcriptional regulators. StbA is organized in two domains. Its N-terminal domain carries the specific stbS DNA binding activity. A truncated version of StbA, deleted of its C-terminal domain, displays only partial activities in vivo, indicating that the non-conserved C-terminal domain is required for efficient segregation and subcellular plasmid positioning. The structure of StbA DNA-binding domain also provides some insight into how StbA monomers cooperate to repress transcription by binding to the stbDR and to form the segregation complex with stbS.


title: Dispersal between interconnected patches can reduce the total population size

abstract: Human activities increasingly result in a fragmentation of natural ecosystems. However, the ecological consequences of fragmentation remain poorly understood. While some studies report that fragmentation may enhance population growth, others suggest the opposite pattern. Here we investigated how habitat connectivity affects the population size of a single species when habitat patches differ in quality. We combined dispersal experiments, in which bacterial populations of Escherichia coli were repeatedly transferred between two qualitatively different environments, with a process-based mathematical model. Both experiments and model consistently revealed that increased dispersal between patches reduced the total population size, thus demonstrating a detrimental effect of habitat connectivity on population size. This observation could be explained with a net loss of individuals upon migration from a productive to an overcrowded patch. Our findings suggest that conservation measures, which promote movement between fragmented habitats, such as dispersal corridors or stepping stones, are potentially detrimental for some species.


title: Adaptation of the eyes of grass puffer (Takifugu niphobles) to the riverine and marine environments

abstract: Since fish inhabit heterogeneous light environments, fish vision is known as a prime example of adaptive evolution. Many species of marine fish migrate to rivers, but little is known about whether these species switch their vision when inhabiting rivers or whether they are adapted to the environment of both rivers and the sea. Grass puffer (Takifugu niphobles) is a marine fish species that frequently migrate to the rivers. In this study, we investigate grass puffers from both riverine and marine populations and analyzed the gene expression in the eyes. Phylogeny and population genetics of riverine and marine grass puffers indicated that riverine and marine grass puffers are from the same population. High-throughput RNA sequencing was used to compare expression levels of two marines and one riverine pufferfish species, as well as riverine and marine glass puffers. The results suggest that the grass puffers are estimated to be adapting their visual system to two environments, the river and the sea, rather than switching their visual system by changing the expression of vision-related genes. Four of the six genes with higher expression in riverine populations than in marine populations of grass puffer were heat shock proteins and related genes. The expression levels of these genes were higher in riverine grass puffer than in other marine and river pufferfish species, suggesting that the grass puffer individuals are adapting to the environmental difference that they face when they migrate to the river by increasing the expression levels of heat shock protein and related genes.

category: evolutionary biology


title: The mutational landscape of single neurons and oligodendrocytes reveals evidence of inflammation-associated DNA damage in multiple sclerosis

abstract: Neuroinflammation has been linked to DNA damage in multiple sclerosis (MS), but its impact on neural cell genomes at nucleotide resolution is unknown. To address this question, we performed single nucleus whole genome sequencing to determine the landscape of somatic mutation in 172 neurons and oligodendrocytes (OLs) extracted from post-mortem brain tissue from 5 MS cases and three controls. We identified two cases with a significant excess of somatic single nucleotide variants (sSNV) in neurons and OLs from MS inflammatory demyelinated lesions. For a case with primary progressive MS, this translated to a 68% increase in sSNV frequency and 32-year equivalent increase in biological age of lesion-resident cells. Mutational signature analysis conducted on all cells revealed that defective DNA repair and transcription-associated DNA damage are important mutagenic mechanism in both neurons and OLs in MS. Our findings provide the first evidence that inflammation in the brains of people with MS is associated with DNA damage, which may have implications for other neurodegenerative diseases and future drug development.

category: genomics


title: A chromosome-length genome assembly and annotation of blackberry (Rubus argutus, cv. Hillquist)

abstract:
BackgroundBlackberries (Rubus spp.) are the fourth most economically important berry crop worldwide. Genome assemblies and annotations have been developed for Rubus species in subgenus Idaeobatus, including black raspberry (R. occidentalis), red raspberry (R. idaeus), and R. chingii, but very few genomic resources exist for blackberries and their relatives in subgenus Rubus.

FindingsHere we present a chromosome-length assembly and annotation of the diploid blackberry germplasm accession  Hillquist (R. argutus).  Hillquist is the only known source of primocane-fruiting (annual-fruiting) in tetraploid fresh-market blackberry breeding programs and is represented in the pedigree of many important cultivars worldwide. The  Hillquist assembly, generated using PacBio long reads scaffolded with Hi-C sequencing, consisted of 298 Mb, of which 270 Mb (90%) was placed on seven chromosome-length scaffolds with an average length of 38.6 Mb. Approximately 52.8% of the genome was composed of repetitive elements. The genome sequence was highly collinear with a novel maternal haplotype-resolved linkage map of the tetraploid blackberry selection A-2551TN and genome assemblies of R. chingii and red raspberry. A total of 38,503 protein-coding genes were predicted using the assembly and Iso-Seq and RNA-seq data, of which 72% were functionally annotated.

ConclusionsThe utility of the  Hillquist genome has been demonstrated here by the development of the first genotyping-by-sequencing based linkage map of tetraploid blackberry and the identification of several possible candidate genes for primocane-fruiting within the previously mapped locus. This chromosome-length assembly will facilitate future studies in Rubus biology, genetics, and genomics and strengthen applied breeding programs.


title: Inferring sources of suboptimality in perceptual decision making using a causal inference task

abstract: Perceptual decision-making has been extensively modeled using the ideal observer framework. However, a range of deviations from optimality demand an extension of this framework to characterize the different sources of suboptimality. Prior work has mostly formalized these sources by adding biases and variability in the context of specific process models but are hard to generalize to more complex tasks. Here, we formalize suboptimalities as part of the brains probabilistic model of the task. Data from a traditional binary discrimination task cannot separate between different kinds of biases, or between sensory noise and approximate computations. We showed that this was possible using a recently developed causal inference task in which observers discriminated auditory cues in the presence of choice-uninformative visual cues. An extension of the task with different stimulus durations provided evidence for an increase in the precision of the computations with stimulus duration, separate from a decrease in observation noise.

category: animal behavior and cognition


title: Protein complex prediction using Rosetta, AlphaFold, and mass spectrometry covalent labeling

abstract:
Structural mass spectrometry offers several techniques for the characterization of protein structures. Covalent labeling (CL) in combination with mass spectrometry can be used as an analytical tool to study and determine structural properties of protein-protein complexes. Degrees of modification obtained from CL experiments for specific labeled residues can be compared between the unbound and bound states of complexes. This analysis can yield insights into structural features of these protein assemblies, specifically the proximity of specific residues to the protein-protein interface. However, this data is sparse and does not unambiguously elucidate protein structure. Thus, computational algorithms are needed to deduce structure from the CL data. In this work we present a novel hybrid method that combines models of protein complex subunits generated with AlphaFold with differential CL data via a CL-guided protein-protein docking in Rosetta. In a benchmark set, the RMSD (root-mean-square deviation) of the best-scoring models was below 3.6 [A] for 5/5 complexes with inclusion of CL data, whereas the same quality was only achieved for 1/5 complexes without CL data. The average improvement in RMSD observed upon inclusion of CL data was 5.2 [A]. This study suggests that our integrated approach can successfully use data obtained from CL experiments to distinguish between nativelike and non-nativelike models.

Significance StatementStructural mass spectrometry can be a powerful and versatile approach to characterize the structure of protein complexes. Data obtained from covalent labeling mass spectrometry can provide insights into higher order protein structure (particularly with respect to residue interactions and solvent accessibility) but needs to be supplemented by computational techniques to elucidate accurate, atomic-detail structural information. Here, we present a method to combine bioanalytical data obtained from covalent labeling with models generated using AlphaFold to accurately predict protein-protein complexes in Rosetta. Differential covalent labeling data can be used to determine the proximity of residues to the binding interface of complexes which we utilized to analyze computational models and improve structure prediction algorithms.

category: biophysics


title: MEK inhibitor resistance in lung cancer cells associated with addiction to sustained ERK suppression

abstract: MEK inhibitors have yielded limited efficacy in KRAS-mutant lung adenocarcinoma (LUAD) patients due to drug resistance. We established trametinib-resistant KRAS-mutant LUAD cells and describe a state of "drug addiction" in a subset of resistant cases where cells are dependent on trametinib for survival. Dependence on ERK2 suppression underlies this phenomenon whereby trametinib removal hyperactivates ERK and results in ER stress and apoptosis. Amplification of KRASG12C occurs in drug-addicted cells and blocking mutant specific activity with AMG 510 rescues the lethality after trametinib withdrawal. Furthermore, increased KRASG12C expression is lethal to other KRAS mutant LUAD cells, consequential to ERK hyperactivation. Our study represents the first instance of this phenotype associated with KRAS amplification and demonstrates that acquired genetic changes that develop in the background of MAPK suppression can have unique consequence. We suggest that the presence of mutant KRAS amplification in patients may identify those that may benefit from a "drug holiday" to circumvent drug resistance. These findings demonstrate the toxic potential of hyperactive ERK signaling and highlight potential therapeutic opportunities in patients bearing KRAS mutations.

category: cancer biology


title: Effects of lowered o and membrane depolarization on the Ca2+ transients of fast skeletal muscle fibers. Implications for muscle fatigue.

abstract:
Sodium (Na+) and potassium (K+) movements during repetitive stimulation of skeletal muscle fibers leads to lowered transmembrane Na+ and K+ gradients. Impaired calcium release resulting from the predicted reduction of the action potential (AP) overshoot (OS) has been suggested as a causative factor of muscle fatigue.

To test this hypothesis, we used a double grease-gap method and simultaneously recorded membrane action potentials (MAPs) and Ca2+ release (as Ca2+ transients), elicited by single pulses or short trains of pulses (100 Hz, 100 ms), in rested fibers polarized to membrane potentials (Vm) between -100 to -55 mV, and exposed to various extracellular Na+ concentrations ([Na+]o; 115, 90, 60 and 40 mM).

In single stimulation experiments, we found that at physiological Vm (-100 mV), Ca2+ release was mostly immune to [Na+]o reductions up to 60 mM (~1/2 the physiological value). In contrast, at 40 mM Na+o Ca2+ release was reduced by 80%, notwithstanding robust MAPs with large OS (~30 mV) were recruited in this conditions.

At Vm between -100 and -60 mV, a 20% reduction of [Na+]o (115 to 90 mM) had no major detrimental effects on Ca2+ release. Instead, depolarization-dependent potentiation of Ca2+ transients, with a maximum at -65 mV, was observed at both 115 and 90 mM Na+o. Potentiation was smaller at 90 mM Na+o. At both [Na+]o, maximally potentiated Ca2+ transients (i.e. at -60 mV) were recruited by MAPS with reduced OSs.

In contrast, Ca2+ release was significantly depressed and no potentiation was observed at Vm between -100 to -70 mV when [Na+]o was reduced 60 mM.

At extreme Na+o (40 mM), Ca2+ release recorded at Vm between -100 and -70 mV was almost obliterated; nonetheless robust MAPs, with OSs of ~25 mV, were recruited.

Extreme depolarizations significantly depressed Ca2+ release at all [Na+]o tested. The Vm leading to Ca2+ release depression was more negative the lower the [Na+]o (-55, -60 and -70 for 115, 90 and mM Na+o, respectively).

Fiber exposed to 115-60 mM Na+o can sustain normal Ca2+ release at a frequency of 100 Hz when polarized between -100 and -80 mV. Depolarizations beyond -80 mV lead to impaired Ca2+ release along the trains. In most cases, there was no correlation between changes in Ca2+ release and changes in OS. At 40 mM Na+o, only the 1st-3rd stimuli of trains recruited Ca2+ transients, which were significantly depressed vis a vis close to normal MAPs.

Neither the OS nor the duration of MAPs are figures of merit predicting the amplitude of Ca2+ transients. At critical combinations of depolarization, [Na+]o, and stimulation frequency, potentiated Ca2+ transients are recruited by MAPS with small OSs; and conversely, partial or total decoupling of Ca2+ release from close to normal MAPs was observed.

Depolarization and Na+o deprivation depressed Ca2+ release in a synergistic way; lowered [Na+]o increased the detrimental effects of depolarization on Ca2+ release, and depolarization render the ECC process more sensitivity to Na+o deprivation.

Impaired TTS AP generation and/or conduction may explain the detrimental effects of depolarization and Na+o deprivation on Ca2+ release.

The effects of increased K+o and Na+o deprivation on the force generation of rested fibers can be explained on the basis of the effects of membrane depolarization and Na+o deprivation on Ca2+ release.

Definitions[ion]i, [ion]o: intracellular and extracellular ion concentrations; ion= Na+, K+, Ca+2. (in molar units)

EFM-Na, EMF-K: electromotive force of Na+ and K+ (in mV)

ENa, EK: equilibrium potential for Na+ and Na+ (in mV)

Vm: membrane or holding potential (in mV)

TTS: transverse tubular system.

Ca-FWHM, Ca+2 transient full-width at half-maximum (in ms)

MAP-FWHM: MAP full-width at half-maximum (in ms)

REF: releasing effective time, time a MAP waveform is above -40 mV (in ms)


title: NSF/αSNAP2-mediated cis-SNARE complex disassembly precedes membrane fusion generating the cell plate during Arabidopsis cytokinesis

abstract: Eukaryotic membrane fusion requires trans-SNARE complexes bridging the gap between adjacent membranes (Jahn and Scheller, 2006). Fusion between a transport vesicle and its target membrane transforms the trans-into a cis-SNARE complex. The latter interacts with the hexameric AAA+-ATPase NSF and its co-factor SNAP, forming a 20S complex (Zhou et al., 2015; Zhao and Brunner, 2016). ATPase activity disassembles the SNARE complex into Qa-SNARE, which folds back onto itself, and its partners (Huang et al., 2019; Kim et al., 2021). Fusion of identical membranes has a different sequence of events (Baker and Hughson, 2016). The fusion partners each have cis-SNARE complexes to be broken up by NSF and SNAP. The Qa-SNARE monomers are then stabilized by interaction with Sec1-type regulators (SM proteins) to form trans-SNARE complexes, as shown for the yeast vacuole (Baker et al., 2015). Membrane fusion in Arabidopsis cytokinesis is formally akin to vacuolar fusion (Muller and Jurgens, 2016). Membrane vesicles fuse with one another to form the partitioning membrane known as cell plate. Cis-SNARE complexes of cytokinesis-specific Qa-SNARE KNOLLE and its SNARE partners are assembled at the ER and delivered by traffic via Golgi/TGN to the cell division plane (Karnahl and Park et al., 2017). SM protein KEULE is required for the formation of trans-SNARE complexes between adjacent membrane vesicles (Park et al., 2012). Here, we identify the missing NSF-type AAA+-ATPase and its adaptor SNAP2 required for disassembly of KNOLLE cis-SNARE complexes. In addition, we show that NSF is also required for other trafficking pathways and interacts with the respective Q-SNAREs. In conclusion, the SNARE complex disassembly machinery is conserved in plants and plays a unique essential role in cytokinesis.


title: Chemical ecology of Himalayan eggplant variety's antixenosis: Identification of geraniol as an oviposition deterrent against the eggplant shoot and fruit borer

abstract:
O_LIEggplant (Solanum melongena) suffers severe losses due to a multi-insecticide resistant lepidopteran pest, shoot and fruit borer (SFB, Leucinodes orbonalis). Heavy and combinatorial application of pesticides for SFB control renders eggplant risky for human consumption.
C_LIO_LIWe observed that 1) ovipositing SFB females can find even solitary plants of susceptible varieties and 2) they do not oviposit on Himalayan eggplant variety RC-RL-22 (RL22). We hypothesized that the olfactory cues influence ovipositing females host choice.
C_LIO_LITo find these cues, leaf volatile blends of seven eggplant varieties were profiled using GCMS. Seven compounds were present in >2.5-fold concentrations in RL22 than the other varieties. In choice assays, oviposition deterrence efficacies of these candidate compounds were independently tested by their foliar application on SFB-susceptible varieties. Complementation of geraniol, which was exclusively found in RL22, reduced oviposition (>90%). To validate geraniols role in RL22s SFB-deterrence, we silenced RL22s geraniol synthase gene using virus-induced gene silencing. Geraniol biosynthesis suppression rendered RL22 SFB-susceptible; foliar geraniol application on the geraniol synthase-silenced plants restored oviposition deterrence.
C_LIO_LIWe infer that geraniol is RL22s SFB oviposition deterrent. The use of natural compounds like geraniol, which influence the chemical ecology of oviposition can reduce the load of hazardous larvicidal pesticides.
C_LI


title: Local field potentials and single unit dynamics in motor cortex of unconstrained macaques during different behavioral states

abstract:
Different sleep states have been shown to be vital for a variety of brain function, including learning, memory, and skill consolidation. However, our understanding of neural dynamics during sleep and the role of prominent LFP frequency bands remain incomplete. To discern changes between different behavioral states we collected multichannel LFP and spike data in primary motor cortex of unconstrained macaques for up to 24 hours using the Neurochip3. Each 8 second bin of time was classified into awake and moving (Move), awake and at rest (Rest), REM sleep (REM), or non-REM sleep (NREM) by using dimensionality reduction and clustering on the average spectral density and the acceleration of the head. LFP power showed high delta during NREM, high theta during REM, and high beta when the animal was awake.

Cross-frequency phase-amplitude coupling typically showed higher coupling for deeper sleep between all pairs of frequency bands except for high delta-high gamma and theta-high gamma coupling during Move, and high theta-beta coupling during REM. Sorted single units showed decreased firing rate with deeper sleep, though with higher "bursty" patterns during NREM compared to other states. Spike-LFP synchrony showed high delta synchrony during Move, and higher coupling with all other frequency bands during NREM. These results altogether are consistent with previous findings showing reactivation of cortical circuitry activated during the day, which may be driven by the delta band LFP.


title: EndophilinA-dependent coupling between activity-dependent calcium influx and synaptic autophagy is disrupted by a Parkinson-risk mutation

abstract: Neuronal activity and neurotransmitter release cause use-dependent decline in protein function. However, it is unclear how this is coupled to local protein turnover and quality control mechanisms. Here we show that the endocytic protein Endophilin-A (EndoA/ENDOA1) couples activity-induced calcium influx to synaptic autophagy and neuronal survival. We identify single mutations in the EndoA flexible region that either increases EndoA diffusion and promotes autophagosome formation in the absence of calcium, or immobilizes EndoA and blocks autophagy, even in the presence of calcium. Hence, the EndoA flexible region is a switch that responds to calcium, regulating EndoA nanoscale synaptic organization and association with autophagosomes driving their formation. Interestingly, a pathogenic variant in the human ENDOA1 variable region that confers risk to Parkinsons disease (PD), also confines ENDOA1 to the synaptic plasma membrane and equally blocks autophagy in flies in vivo and in induced human neurons. Thus, our work reveals a mechanism neurons use to connect neuronal activity to local protein turnover by autophagy, which is critical for neuronal survival.


title: Effects of calcium-regulated autophagy on Candidatus Liberibacter solanacearum in carrot psyllid midguts

abstract: Candidatus Liberibacter solanacearum (CLso) transmitted by the carrot psyllid, Bactericera trigonica causes carrot yellows in Israel, and has recently gained much importance due to the excessive economical loss. Understanding the interactions between CLso and the psyllid at the cellular level is fundamental for the disease management. Here, we demonstrate the role of calcium ATPase, cytosolic calcium and most importantly Beclin1 in regulating autophagy and its association with Liberibacter. Presence of CLso generates reactive oxygen species and induces the expression of the detoxification enzymes in the psyllid midguts. CLso also induces the expression of both sarco/endoplasmic reticulum Ca2+ pump (SERCA) and 1,4,5-trisphosphate receptors (ITPR) in the midguts, followed by high levels of calcium in the cytosol. Silencing these proteins individually disrupted the calcium levels in the cytosol leading to direct effects on autophagy and thus on Liberibacter. On the other hand, inhibiting Beclin1-phosphorylation through different calcium induced kinases altered the expression of autophagy and CLso abundance. This study establishes a direct correlation between cytosolic calcium levels, autophagy and CLso in the carrot psyllid midgut.

category: molecular biology


title: MCIBox: A Toolkit for Single-molecule Multi-way Chromatin Interaction Visualization and Micro-Domains Identification

abstract: The emerging ligation-free three-dimensional (3D) genome mapping technologies can identify multiplex chromatin interactions with single-molecule precision. These technologies offer new insight into high-dimensional chromatin organization and gene regulation, but also introduce new challenges in data visualization and analysis. To overcome these challenges, we developed MCIBox, a toolkit for Multi-way Chromatin Interaction (MCI) analysis, including a visualization tool and a platform for identifying micro-domains with clustered single-molecule chromatin complexes. MCIBox is based on various clustering algorithms integrated with dimensionality reduction methods that can display multiplex chromatin interactions at single-molecule level, allowing users to explore chromatin extrusion patterns and super-enhancers regulation modes in transcription, and to identify single-molecule chromatin complexes that are clustered into micro-domains. Furthermore, MCIBox incorporates a two-dimensional kernel density estimation algorithm to identify micro-domains boundaries automatically. These micro-domains were stratified with distinctive signatures of transcription activity and contained different cell cycle associated genes. MCIBox could potentially distinguish the specificity of single-molecule chromatin interaction patterns in various phases of a cell cycle or cell types.


title: Drug-like small molecules that inhibit expression of the oncogenic microRNA-21

abstract: We report the discovery of a series of drug-like small molecules which bind specifically to the precursor of the oncogenic and pro-fibrotic microRNA-21 with mid-nanomolar affinity. These molecules are highly ligand-efficient (MW<330) and display specific biochemical and cellular activity by suppressing maturation of miR-21, thereby providing an avenue towards therapeutic intervention in multiple diseases where miR-21 is abnormally expressed. The small molecules target a local structure at the Dicer cleavage site and induce distinctive structural changes in the RNA which correlate with specific inhibition of miRNA processing. Structurally conservative single nucleotide substitutions eliminate the conformational change, which is not observed in other miRNA precursors. The most potent of these compounds reduces cellular proliferation and miR-21 levels in cancer cell lines without inhibiting kinases or classical receptors, while closely related compounds without this specific binding activity are inactive in cells.

category: biochemistry


title: A novel computational approach to identify cancer cells in scRNA-seq data

abstract: Single-cell RNA-seq is an invaluable research tool that allows for the investigation of gene expression in heterogeneous cancer cell populations in ways that bulk RNA-seq cannot. However, normal (i.e., non tumor) cells in cancer samples have the potential to confound the downstream analysis of single-cell RNA-seq data. Several existing methods for identifying tumor cells use copy number variation inference. This work aims to extend existing approaches for identifying cancer cells in single-cell RNA-seq samples by incorporating putative driver alterations. We found that putative driver alterations can be detected in single-cell RNA-seq data and that a subset of cells in tumor samples are enriched in putative driver alterations as compared to normal cells. Furthermore, we show that the number of putative driver alterations and inferred copy number variation are not correlated in all samples. Taken together, our findings suggest that combining copy number variation inference with putative driver mutation load can augment the number of tumor cells that can be confidently included in downstream analyses of single-cell RNA-seq datasets.


title: Experimental Guidance for Discovering Genetic Networks through Iterative Hypothesis Reduction on Time Series

abstract:
Large programs of dynamic gene expression, like cell cyles and circadian rhythms, are controlled by a relatively small "core" network of transcription factors and post-translational modifiers, working in concerted mutual regulation. Recent work suggests that system-independent, quantitative features of the dynamics of gene expression can be used to identify core regulators. We introduce an approach of iterative network hypothesis reduction from time-series data in which increasingly complex features of the dynamic expression of individual, pairs, and entire collections of genes are used to infer functional network models that can produce the observed transcriptional program. The culmination of our work is a computational pipeline, Iterative Network Hypothesis Reduction from Temporal Dynamics (Inherent Dynamics Pipeline), that provides a priority listing of targets for genetic perturbation to experimentally infer network structure. We demonstrate the capability of this integrated computational pipeline on synthetic and yeast cell-cycle data.

Author SummaryIn this work we discuss a method for identifying promising experimental targets for genetic network inference by leveraging different features of time series gene expression data along a chained set of previously published software tools. We aim to locate small networks that control oscillations in the genome-wide expression profile in biological functions such as the circadian rhythm and the cell cycle. We infer the most promising targets for further experimentation, emphasizing that modeling and experimentation are an*Corresponding author: breschine.cummins@montana.edu essential feedback loop for confident predictions of core network structure. Our major offering is the reduction of experimental time and expense by providing targeted guidance from computational methods for the inference of oscillating core networks, particularly in novel organisms.

category: systems biology


title: Genetic epidemiology of human neutrophil antigen variants suggest significant global variability

abstract: Human neutrophil antigens possess significant clinical implications especially in the fields of transfusion and transplantation medicine. Efforts to estimate the prevalence of genetic variations underpinning the antigenic expression are emerging. However, there lacks a precise capture of the global frequency profiles. Our article emphasizes the potential utility of maintaining an organized online repository of evidence on neutrophil antigen associated genetic variants from published literature and reports. This in our opinion, is an emerging area and would significantly benefit from the awareness and understanding of population-level diversities.


title: INPP5D limits plaque formation and glial reactivity in the APP/PS1 mouse model of Alzheimer's disease

abstract: The dual specificity lipid/protein phosphatase SHIP1 (encoded by the INPP5D gene) is enriched in myeloid cells. Single nucleotide polymorphisms (SNPs) in INPP5D coding and non-coding regions impact risk for developing late onset sporadic Alzheimers disease (LOAD). We present pathological analyses with spatial transcriptomics of mice with tamoxifen-sensitive microglial knockdown of Inpp5d and show exacerbated plaque pathology, plaque-associated microglial density, and altered gene expression around plaques, suggesting novel markers for plaque-associated reactive microglia.


title: Asynchronous excitatory neuron development in an isogenic cortical spheroid model of Down syndrome

abstract: The intellectual disability (ID) in Down syndrome (DS) is thought to result from a variety of developmental deficits such as alterations in neural progenitor division, neurogenesis, gliogenesis, cortical architecture, and reduced cortical volume. However, the molecular processes underlying these neurodevelopmental changes are still elusive, preventing an understanding of the mechanistic basis of ID in DS. In this study, we used a pair of isogenic (trisomic and euploid) induced pluripotent stem cell (iPSC) lines to generate cortical spheroids (CS) that model the impact of trisomy 21 on brain development. CS contain neurons, astrocytes, and oligodendrocytes and they are widely used to approximate early neurodevelopment. Using single cell RNA sequencing (scRNA-seq), we uncovered cell type-specific transcriptomic changes in the trisomic CS. In particular, we found that excitatory neuron populations were most affected and that a specific population of cells with a transcriptomic profile resembling layer IV cortical neurons displayed the most profound divergence in developmental trajectory between trisomic and euploid genotypes. We also identified candidate genes potentially driving the developmental asynchrony between trisomic and euploid excitatory neurons. Direct comparison between the current isogenic CS scRNA-seq data and previously published datasets revealed several recurring differentially expressed genes between DS and control samples. Altogether, our study highlights the power and importance of cell type-specific analyses within a defined genetic background, coupled with broader examination of mixed samples, to comprehensively evaluate cellular phenotypes in the context of DS.


title: Colour polymorphism associated with a gene duplication in male wood tiger moths

abstract: Colour is often used as an aposematic warning signal, with predator learning expected to lead to a single colour pattern within a population. However, there are many puzzling cases where aposematic signals are also polymorphic. The wood tiger moth, Arctia plantaginis, uses bright hindwing colours as a signal of unpalatability, and males have discrete colour morphs which vary in frequency geographically. In Finland, both white and yellow morphs can be found, and these colour morphs also differ in behavioural and life-history traits. Complex polymorphisms such as these are often explained by supergenes. Here, we show that male colour is linked to an extra copy of a yellow family gene that is only present in the white morphs. This white-specific duplication, which we name valkea, is highly upregulated during wing development, and could act to reduce recombination, thus potentially representing a supergene. We also characterise the pigments responsible for yellow, white and black colouration, showing that yellow is partly produced by pheomelanins, while black is dopamine-derived eumelanin. The yellow family genes have been linked to melanin synthesis and behavioural traits in other insect species. Our results add to only a few examples of seemingly paradoxical and complex polymorphisms which are associated with single genes.


title: Comparison of selective genotyping strategies in genomic selection programs for broilers using stochastic simulation

abstract: Breeding programs using genomic information have become common in broilers. In broilers, the main traits for selection are body weight and feed efficiency. These traits are measured in both sexes and before sexual maturity. Thus, increases in genetic gain in broiler breeding programs due to the use of genomic information are attributable primarily to the increased accuracy of predicted breeding values of potential parents. As not all birds can be genotyped due to economic constraints, an optimal genotyping strategy needs to be chosen. The objective of this study was to investigate the rates of genetic gain and inbreeding obtained by varying three factors: the percentage of birds genotyped (PG), the percentage of birds genotyped randomly (PRG) or selectively, and the percentage of males among genotyped birds (PMG). Stochastic computer simulation with a full factorial experimental design was used. Significant interaction among the factors (PG, PRG, and PMG) was observed for the rate of genetic gain in body weight and aggregate breeding value, but not for the gain in residual feed intake or the rate of inbreeding per generation. Our findings indicate that the PG, PRG, and PMG need to be considered when selecting a genotyping strategy for a broiler breeding program. If available resources allow only to genotype a small percentage (e.g., 2.5% or 5%) of all birds, the genotyping of 50-100% of male birds and selection of birds to be genotyped using phenotypic information is best. If resources allow to genotype more (e.g., [≥]20%) candidates, genotyping of equal numbers of each sex, and low PRG level (i.e., preselection of the majority of candidate based on performance) is best. Provided that a proper genotyping strategy is chosen, we conclude that the incorporation of genomic information in broiler breeding programs can substantially increase the rate of genetic gain.

category: genetics


title: Bacterial plasmid-associated and chromosomal proteins have fundamentally different properties in protein interaction networks

abstract:
Plasmid-encoded genes facilitate the spread of antimicrobial resistance (AMR) though horizontal gene transfer (HGT). HGT driven by plasmids enables the diversification of pathogens into new anatomical and environmental niches, implying that plasmid-bourne genes can cooperate well with chromosomal genes. It is hypothesised that such mobile genes are functionally different to chromosomal ones due to this ability to encode non-essential functions like AMR and traverse distinct host cells. The effect of plasmid-related gene gain on protein-protein interaction network topology is an important question in this area. Moreover, the extent to which these chromosomally- and plasmid-encoded proteins interact with proteins from their own groups compared to the levels with the other group remains unclear. Here, we examined the incidence and protein-protein interactions of all known plasmid-linked genes (n=9,172) across representative specimens from all bacteria (n=4,419) using all known unique plasmids. We found that such plasmid-related genes constitute on average of 10.5% of the total number of genes per bacterial sample, and that plasmid genes preferentially associated with different species such that they provide moderate taxonomical power. Surprisingly, plasmid-related proteins had both more direct and indirect protein-protein interactions compared to chromosomal proteins, countering the hypothesis that genes with higher mobility rates should have fewer protein-level interactions. Nonetheless, topological analysis and investigation of the protein-protein interaction networks connectivity and change in the number of independent components demonstrated that the plasmid-related proteins had limited overall impact in most bacterial samples (>94%). This paper assembled extensive data on plasmid-related proteins, their interactions and associations with diverse bacterial specimens that is available for the community to investigate in more detail.

Significance as a bioresource to the communityThis paper outlines the compilation and analysis of bacterial plasmid-encoded genes and protein-level interactions that will be a useful resource for the community. All input data, output data, code and high-resolution figures is available on FigShare and Github. We extracted protein-protein interaction data for all available valid bacterial samples (n=4,419) to differentiate plasmid-related genes and PPIs from chromosomal ones. We compiled all known unique plasmids (n=18,628) to identify 9,172 unique plasmid-related genes using gene name information, which may require refinement is a useful resource for the community because we found that these had some power to resolve known taxonomic differences, suggesting host-gene co-evolution. The co-varying plasmid-related genes and samples are available for more specific testing. We used this data to identify protein-protein interactions involving plasmid proteins per sample, and showed that plasmid-related proteins tended here to have more protein-protein interactions than chromosomal ones. However, this varied across samples, and thus data allows the community to explore features of selected bacteria and/or genes. We identified a small number of samples (n=158) where the plasmid-related proteins had major effects on protein-protein interaction network structure, which is possible to explore more through this bioresource.

Data Summary

O_TBL View this table:
org.highwire.dtl.DTLVardef@17d1e59org.highwire.dtl.DTLVardef@1539b2aorg.highwire.dtl.DTLVardef@1efed56org.highwire.dtl.DTLVardef@c6bb88org.highwire.dtl.DTLVardef@1420d4d_HPS_FORMAT_FIGEXP  M_TBL O_FLOATNOTable 1.C_FLOATNO O_TABLECAPTIONLarge datasets available from this study on FigShare, showing the item, description and the corresponding DOI link. See the Supplementary Data for more. The code associated with this paper is available on Github at https://github.com/downingtim/mobilome_2022 and data files are there at https://figshare.com/projects/Bacteria_mobilome_2022/136720 "Bacteria_mobilome_2022".

C_TABLECAPTION C_TBL


title: Aggression and multimodal signaling in noise in the European robin

abstract:
The effect on anthropogenic noise on acoustic signals is widely studied although the extent to which this is due to short-term flexibility or long-term adaptation is unclear. Anthropogenic noise may disrupt signals used to mediate aggressive interactions, leading to more physical aggression between opponents. One solution to this problem is to switch signaling effort to a less noisy modality (e.g. the visual modality). In the present study we ask whether urban and rural European robins (Erithacus rubecula) use multi-modal signals flexibly in response to anthropogenic noise during territorial defense. We predicted that during simulated intrusions with experimental noise, European robins would increase their physical aggression, respond with more visual threat displays and sing fewer songs, i.e. show a multi-modal shift. We also predicted that urban birds, living in noisier habitats, would be generally more aggressive than rural birds. The results showed that urban birds were more aggressive than rural robins, but an increase in aggression with experimental noise was seen only in the rural birds. Urban birds also used visual signals more often than rural birds. However, birds did not use visual signals more in experimental noise. Instead, both urban and rural robins sang at higher rates under noise conditions, thus increasing signaling effort in the noisy modality. These results point to a complex role of immediate plasticity and longer-term processes in affecting communication during aggressive interactions under anthropogenic noise.

Significance StatementHuman activity has an enormous effect on wildlife, including on their social behavior. Animals living in urban areas often tend to be more aggressive than those living in rural areas, which may be due to urban acoustic noise making communication between individuals more difficult. In a study with a common songbird, the European robin, we investigated the role of urban acoustic noise in aggression and territorial communication. Urban robins were more aggressive than rural robins, and additional noise in the territory increased aggression in rural but not urban robins. Robins increased their singing effort but did not increase visual signals in acoustic noise. These results suggest that noise can indeed make animals behave more aggressively although the effect may depend on how noisy it is already. These results further our understanding of how human-made noise changes animal communication and social behavior.


title: Statistical correction of input gradients for black box models trained with categorical input features

abstract: Gradients of a deep neural networks predictions with respect to the inputs are used in a variety of downstream analyses, notably in post hoc explanations with feature attribution methods. For data with input features that live on a lower-dimensional manifold, we observe that the learned function can exhibit arbitrary behaviors off the manifold, where no data exists to anchor the function during training. This leads to a random component in the gradients which manifests as noise. We introduce a simple correction for this off-manifold gradient noise for the case of categorical input features, where input values are subject to a probabilistic simplex constraint, and demonstrate its effectiveness on regulatory genomics data. We find that our correction consistently leads to a significant improvement in gradient-based attribution scores.


title: Identification of Key Genes and Biological Pathways in Bipolar Disorder by Bioinformatics and Next Generation Sequencing Data Analysis

abstract: Bipolar disorder (BD), also known as psychiatric disorder, affects millions of people all over the world. The aim of this investigation was to screen and verify hub genes involved in BD as well as to explore potential molecular mechanisms. The next generation sequencing (NGS) dataset GSE124326 was downloaded from the Gene Expression Omnibus (GEO) database, which contained 480 samples, including 240 BD and 240 normal controls. Differentially expressed genes (DEGs) were filtered and subjected to gene ontology (GO) and pathway enrichment analyses. A protein-protein interaction (PPI) network and module analysis were used to identify hub genes. The miRNA-hub gene regulatory network and TF-hub gene regulatory network were also constructed. Receiver operating characteristic curve (ROC) analysis was used to validate hub genes. In this work, 957 DEGs, including 477 up regulated and 480 down regulated, were obtained from NGS data. The GO and pathway enrichment analyses of the DEGs showed that the up regulated genes were enriched in the neutrophil degranulation, immune system, transport, cytoplasm and enzyme regulator activity, and the down regulated genes were enriched in extracellular matrix organization, diseases of metabolism, multicellular organismal process, cell periphery and metal ion binding. Finally, through analyzing the PPI network, miRNA-hub gene regulatory network and TF-hub gene regulatory network, we screened hub genes UBB, UBE2D1, TUBA1A, RPL11, RPS24, NOTCH3, CAV1, CNBD2, CCNA1 and MYH11 by the Cytoscape software. The current investigation illustrates a characteristic NGS data in BD, which might contribute to the interpretation of the progression of BD and provide novel biomarkers and therapeutic targets for BD.


title: Deep Learning-based Pseudo-Mass Spectrometry Imaging Analysis for Precision Medicine

abstract: Liquid chromatography-mass spectrometry (LC-MS) based untargeted metabolomics provides systematic profiling of metabolic. Yet its applications in precision medicine (disease diagnosis) have been limited by several challenges, including metabolite identification, information loss, and low reproducibility. Here, we present the deepPseudoMSI project (https://www.deeppseudomsi.org/), which converts LC-MS raw data to pseudo-MS images and then processes them by deep learning for precision medicine, such as disease diagnosis. Extensive tests based on real data demonstrated the superiority of deepPseudoMSI over traditional approaches and the capacity of our method to achieve an accurate individualized diagnosis. Our framework lays the foundation for future metabolic-based precision medicine.


title: Covalent Docking and Molecular Dynamics Simulations Reveal the Specificity-Shifting Mutations Ala237Arg and Ala237Lys in TEM Beta-Lactamase

abstract:
The rate of modern drug discovery using experimental screening methods still lags behind the rate at which pathogens mutate, underscoring the need for fast and accurate predictive simulations of protein evolution. Multidrug-resistant bacteria evade our defenses by expressing a series of proteins, the most famous of which is the 29-kilodalton enzyme, TEM {beta}-lactamase. Considering these challenges, we applied a covalent docking heuristic to measure the effects of all possible alanine 237 substitutions in TEM due to this codons importance for catalysis and effects on the binding affinities of commercially-available {beta}-lactam compounds. In addition to the usual mutations that reduce substrate binding due to steric hindrance, we identified two distinctive specificity-shifting TEM mutations, Ala237Arg and Ala237Lys, and their respective modes of action. Notably, we discovered and verified through minimum inhibitory concentration assays that, while these mutations and their bulkier side chains lead to steric clashes that curtail ampicillin binding, these same groups foster salt bridges with the negatively-charged side-chain of the cephalosporin cefixime, widely used in the clinic to treat multi-resistant bacterial infections. To measure the stability of these unexpected interactions, we used molecular dynamics simulations and found the binding modes to be stable despite the application of biasing forces. Finally, we found that both TEM mutants also bind strongly to other drugs containing negatively-charged R-groups, such as carumonam and ceftibuten. As with cefixime, this increased binding affinity stems from a salt bridge between the compounds negative moieties and the positively-charged side chain of the arginine or lysine, suggesting a shared mechanism. In addition to reaffirming the power of using simulations as molecular microscopes, our results can guide the rational design of next-generation {beta}-lactam antibiotics and bring the community closer to retaking the lead against the recurrent threat of multidrugresistant pathogens.

Author SummaryResistance to antibiotics is a major public health threat. Microorganisms are able to resist commonly used drugs by evolving and expressing enzymes capable of neutralizing antibiotics. Understanding the relationships between structural elements in these enzymes and their drug-clearing functions can lead to crucial insights for the discovery of next-generation antibiotics. In this study, we have used cutting-edge computational modeling methods to predict the effects of all naturally-occurring variations of an important region of the binding site of TEM {beta}-lactamase, one of the major resistance-granting enzymes in bacteria. In an effort to identify patterns that could be useful for drug discovery, our simulations sought to understand how chemical changes in the tested region can affect resistance against a collection of over 90 widely used antibiotics. Crucially, through our simulations, we have identified a pathway for bacterial resistance against {beta}-lactam antibiotics containing a negatively-charged moiety. We have also elucidated the mechanism behind the gain of resistance, which involves strong interactions between the drugs negative moieties and the positively-charged chemical shifts resulting from the mutation. Finally, we validated our predictions against fitness experiments for two commonly used antibiotics, which qualitatively corroborated our most important findings.


title: Tensor decomposition and principal component analysis-based unsupervised feature extraction outperforms state-of-the-art methods when applied to histone modification profiles

abstract: Identification of histone modification from datasets that contain high throughput sequencing is difficult. Although multiple methods have been developed to identify histone modification, most of these methods are not specific for histone modification, but are general methods that aim to identify protein binding to the genome. In this study, tensor decomposition and principal component analysis based unsupervised feature extraction with optimized standard deviation, which were successfully applied to gene expression and DNA methylation, were used to identify histone modification. The proposed method identified various histone modifications successfully and also outperformed various state-of-the-art methods. The proposed method is expected to be a de facto standard method to identify histone modification.


title: microbeSEG: Accurate Cell Segmentation with OMERO Data Management

abstract: In biotechnology, cell growth is one of the most important properties for the characterization and optimization of microbial cultures. Novel live-cell imaging methods are leading to an ever better understanding of cell cultures and their development. The key for analyzing acquired data is accurate and automated cell segmentation at the single-cell level. Therefore, we present microbeSEG, a user-friendly cell segmentation tool with OMERO data management. microbeSEG utilizes a state-of-the-art deep learning-based segmentation method and can be used for instance segmentation of a wide range of cell morphologies and imaging techniques, e.g., phase contrast or fluorescence microscopy. The main focus of microbeSEG is a comprehensible, easy, efficient, and complete workflow from creation of training data to final application of the trained segmentation model. We demonstrate that accurate cell segmentation results can be obtained within 45 minutes of user time. Utilizing public segmentation data sets or pre-labeling further accelerates the microbeSEG workflow. This opens the door for accurate and efficient data analysis of microbial cultures.


title: The effect of external lateral stabilization on ankle moment control during steady-state walking

abstract: External lateral stabilization can help identify stability control mechanisms during steady-state walking. The degree of step-by-step foot placement control and step width are known to decrease when walking with external lateral stabilization. Here, we investigated the effect of external lateral stabilization on ankle moment control in healthy participants. Ankle moment control complements foot placement, by allowing a corrective center-of-pressure shift once the foot has been placed. This is reflected by a model predicting this center-of-pressure shift based on the preceding foot placement error. Here, the absolute explained variance accounted for by this model decreased when walking with external lateral stabilization. In other words, we found a reduction in the contribution of step-by-step ankle moment control to mediolateral gait stability when externally stabilized. Concurrently, foot placement error and the average center-of-pressure shift remained unchanged.


title: Hypoxia-mediated regulation of DDX5 through decreased chromatin accessibility and post-translational targeting restricts R-loop accumulation

abstract: Local hypoxia (low oxygen) occurs in most solid tumors and is associated with aggressive disease and therapy resistance. Widespread changes in gene expression play a critical role in the biological response to hypoxia. However, most of the prior research has focused on hypoxia-inducible genes in hypoxia as opposed to those which are decreased. Using ATACseq, we demonstrate that chromatin accessibility is decreased in an oxygen-dependent manner, predominantly at gene promoters and specific pathways are impacted including DNA repair, splicing and the R-loop interactome. As R-loops accumulate in hypoxic conditions we hypothesized that an underlying mechanism could be the repression of the R-loop interactome. One of the genes with decreased chromatin accessibility in hypoxia was DDX5, encoding the RNA helicase, DDX5, which showed reduced expression in various cancer cell lines in hypoxic conditions, tumor xenografts and in patient samples with hypoxic tumors. In addition, we identified TRIM5 as a novel hypoxia inducible E3 ligase which targets DDX5 for proteasomal degradation independently of changes at mRNA levels. We demonstrate multiple mechanisms contributing to reduced DDX5 expression in hypoxic conditions. Most interestingly, we found that when DDX5 is rescued in hypoxia, R-loop levels accumulate further, therefore demonstrating that hypoxia-mediated repression of DDX5 restricts R-loop accumulation. Together these data support the hypothesis that a critical part of the biological response to hypoxia is the repression of multiple R-loop processing factors, however, as shown for DDX5, their role is specific and distinct.


title: Non-Invasive Cancer Detection in Canine Urine through C. elegans Chemotaxis

abstract: Cancer is the leading cause of death of companion animals, and successful early treatment has been a challenge in the veterinary field. We have developed the Non-Invasive Cancer Screening (N.C.S.) Study to perform cancer detection through the analysis of canine urine samples. The test makes use of the strong olfactory system of the nematode C. elegans, which was previously shown to positively respond to urine samples from human cancer patients. We performed a proof-of-concept study to optimize the detection capability in urine samples obtained from dogs with naturally occurring cancers. In this study, we established a scale for identifying the cancer risk based on the magnitude of the chemotaxis index of C. elegans towards a canine urine sample. Through validation, the N.C.S. Study achieved a sensitivity of 85%, showing that it is highly sensitive to indicating the presence of cancer across multiple types of common canine cancers. The test also showed a 93% specificity to cancer samples, indicating a low rate of over-identifying cancer risk. From these results, we have demonstrated the ability to perform low-cost, non-invasive cancer detection in companion animals, a method which can increase the ability to perform cancer diagnosis and treatment.


title: Quantum pBac: An effective, high-capacity piggyBac-based gene integration vector system for unlocking gene therapy potential

abstract:
Recent advances in gene therapy have brought novel treatment options to multiple fields of medicine, including cancer. However, safety concerns and limited payload capacity in commonly-utilized viral vectors prevent researchers from unlocking the full potential of gene therapy. Virus-free DNA transposons, including piggyBac, have been shown to obviate these shortcomings. We have previously demonstrated the superior transposition efficiency of a modified piggyBac system in HEK293 cells. Here, we further advanced and broadened the therapeutic application of this modified piggyBac system. We demonstrated that the internal domain sequence (IDS) within the 3 terminal repeat domain of hyperactive piggyBac (hyPB) donor vector contain dominant enhancer elements. We showed that a plasmid-free donor vector having IDS-free terminal inverted repeats in conjunction with a helper plasmid expressing Quantum pBase v2 form the most optimal piggyBac system, Quantum pBac (qPB), in T cells. We further demonstrated that T cells transfected with qPB expressing CD20/CD19 CAR outperformed cells transfected with the same donor vector but with plasmid expressing hyPB transposase in CAR-T cell production. Importantly, we showed that qPB produced mainly CD8+ CAR-T cells that are also highly represented by TSCM. These CAR-T cells effectively eliminated CD20/CD19-expressing tumor cells in vitro and in Raji-bearing immunodeficient mice. Our findings confirm that qPB is a promising virus-free vector system that is safer, and highly efficient in mediating transgene integration with the payload capacity to incorporate multiple genes.

Significance StatementAn effective, high capacity and safe gene integration vector system is critical to the success of gene therapy. We have previously modified a virus-free piggyBac transposon vector system and demonstrated its potential application in gene therapy. Here we further demonstrate that shortening of the piggyBac donor vector terminal repeat domain and backbone, coupled with Quantum pBase (qPBase) v2, result in the most optimal Quantum pBac (qPB) piggyBac system in human T cells. qPBase v2 outperformed hyperactive piggyBac transposase in chimeric antigen receptor T (CAR-T) production. qPB produced CAR-T cells that are mainly CD8+, highly represented by TSCM, and effectively eliminated tumors in vivo. Our findings solidify qPB as a promising virus-free vector system for therapeutic application.


title: A new highly-specific Natural Killer cell-specific gene signature predicting recurrence in colorectal cancer patients.

abstract: The protective role of Natural Killer (NK) cell tumour immunosurveillance has long been recognised in colorectal cancer (CRC). However, as most patients show limited intra-tumoral NK cell infiltration, improving our ability to identify those with high NK cell activity might aid in dissecting the molecular features which could trigger strong response to NK cell-mediated immune killing. Here, a novel CRC-specific NK cell gene signature capable of inferring NK cell load in primary tissue samples was derived and validated in multiple patient CRC cohorts. The specificity of the signature is substantiated in tumour-infiltrating NK cells from primary CRC tumours at the single cell level, and the expression profile of each constituent gene is explored in NK cells of different maturation states, activation status and anatomical origin. Thus, in contrast with other NK cell gene signatures that have several overlapping genes across different immune cell types, our NK cell signature has been extensively refined to be specific for CRC-infiltrating NK cells and includes genes which identify a broad spectrum of NK cell subtypes. Moreover, it was shown that this novel NK cell signature accurately discriminates murine NK cells, demonstrating the potential applicability of this signature when mining datasets generated from both clinical and animal studies. Differential gene expression analysis revealed tumour-intrinsic features associated with NK cell inclusion versus exclusion in CRC patients, with those tumours with predicted high NK activity showing strong evidence of enhanced chemotactic and cytotoxic transcriptional programs. Furthermore, survival modelling indicated that NK signature expression is associated with improved survival outcomes in two large cohorts of primary CRC patients. Thus, scoring CRC samples with this refined NK cell signature might aid in identifying patients with high NK cell activity who could be prime candidates for NK cell directed immunotherapies.


title: Uncovering Additional Predictors of Urothelial Carcinoma from Voided Urothelial Cell Clusters Through a Deep Learning Based Image Preprocessing Technique

abstract: Urine cytology is commonly used as a screening test for high grade urothelial carcinoma for patients with risk factors or hematuria and is an essential step in longitudinal monitoring of patients with a prior bladder cancer history. However, the semi-subjective nature of current reporting systems for urine cytology (e.g., The Paris System) can hamper reproducibility. For instance, the incorporation of urothelial cell clusters into the classification schema is still an item of debate and perplexity amongst expert cytopathologists, as several previous works have disputed their diagnostic relevance. Recently, several machine learning and morphometric algorithms have been proposed to provide quantitative descriptors of urine cytology specimens in an effort to reduce subjectivity and include automated assessments of cell clusters. However, it remains unclear how these computer algorithms interpret/analyze cell clusters. In this work, we have developed an automated preprocessing tool for urothelial cell cluster assessment that divides urothelial cell clusters into meaningful components for downstream assessment (i.e., population-based studies, workflow automation). Results indicate that cell cluster atypia (i.e., defined by whether the cell cluster harbored multiple atypical cells, thresholded by a minimum number of cells), cell border overlap and smoothness, and total number of clusters are important markers of specimen atypia when considering assessment of urothelial cell clusters. Markers established through techniques to separate cell clusters may have wider applicability for the design and implementation of machine learning approaches for urine cytology assessment.

category: pathology


title: Characterization of metabolic changes associated with floral transition in Arabidopsis: RAFFINOSE SYNTHASE 5 contributes to determine the timing of floral transition

abstract: Integration of environmental and endogenous cues triggers floral induction at the optimal time during the plant life cycle. Flowering is a tightly regulated process, which involves an intricated genetic network, as expected for a process crucial for plant fitness and survival. Individual metabolites are known to contribute to the determination of flowering time, including carbohydrates and hormones. However, a global analysis of metabolic changes associated with flowering was still lacking. We performed a metabolomic study to characterize global metabolic changes associated with photoperiodic floral induction. By using an inducible system, with the CONSTANS (CO) promoter driving the expression of CO fused to the rat glucocorticoid receptor (CO::GR), we induce flowering and identify metabolites that increase or decrease in leaves and apices during floral induction. Combining metabolomic with transcriptomic data, we identify that raffinose metabolism was altered in apices that are induced to flower. Loss-of-function mutants affecting RAFFINOSE SYNTHASE 5 (RS5), a key enzyme of the raffinose metabolism, show an early flowering phenotype. Also, RS5 expression changes during floral transition, suggesting a role for raffinose catabolism on the release of simple sugars at the apex. We propose that variation on the differential accumulation of raffinose and mono- and disaccharides during floral transition contributes to the induction of floral transition, by influencing expression of THEHALOSE-6-PHOSPHATE SYNTHASE 1 (TPS1) and SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 3 (SPL3), which affect expression of the florigen FLOWERING LOCUS T (FT).

category: plant biology


title: Leaf-level metabolic changes in response to drought affect daytime CO2 emission and isoprenoid synthesis

abstract:
Daytime respiration in leaves is an integral component of plant metabolism, with CO2 produced as a byproduct during the synthesis of many secondary metabolites, including many common volatile organic compounds (VOCs). It remains unclear how the carbon fluxes associated with daytime respiration and VOC emission vary among mature trees, and how they are influenced by drought. We used position-specific 13C-pyruvate labeling to investigate CO2 and VOC fluxes from the leaves of four tropical species before and during an artificially imposed drought in an enclosed rainforest. Although daytime respiration was always dominated by non-mitochondrial processes, the relative contribution of CO2 from the tricarboxylic acid cycle increased under drought, likely due to osmolyte production. In the legume tree Clitoria fairchildiana, our data indicate that intermediates from the mevalonic acid pathway are used to produce the volatile monoterpene trans-{beta}-ocimene, but not isoprene. This suggests that geranyl pyrophosphate is the more important intermediate for crosstalk between cytosolic and plastidic isoprenoid synthesis, not isopentenyl pyrophosphate. Crosstalk declined with drought, and although trans-{beta}-ocimene emissions increased, they were primarily sourced from storage and not de novo synthesis. We found highly species-specific dynamics in carbon allocation into different metabolic processes including daytime respiration and VOC synthesis.

HighlightIntracellular carbon allocation into daytime respiration and VOCs is species-specific and changes with drought. Isoprenoid synthesis in the plastid can be fed with glyceraldehyde-3-phosphate and geranyl pyrophosphate from the cytosol.


title: Taxonomically Restricted Genes are Associated with Responses to Biotic and Abiotic Stresses in Sugarcane (Saccharum spp.)

abstract: Orphan genes (OGs) are protein-coding genes that are restricted to particular clades or species and lack homology with genes from other organisms, making their biological function difficult to predict. OGs can rapidly originate and become functional; consequently, they may support rapid adaptation to environmental changes. Extensive spread of mobile elements, and whole genome duplication, occurred in the Saccharum group, which may have contributed to the origin and diversification of OGs in the sugarcane genome. Here, we identified and characterized OGs in sugarcane, examined their expression profiles across tissues and genotypes, and investigated their regulation under varying conditions. We identified 319 OGs in the Saccharum spontaneum genome without detected homology to protein-coding genes in green plants, except those belonging to Saccharinae. Transcriptomic analysis showed 288 sugarcane OGs with detectable expression levels in at least one tissue or genotype. We observed similar expression patterns of OGs in sugarcane genotypes originating from the closest geographical locations. We also observed tissue-specific expression of some OGs, possibly indicating a complex regulatory process for maintaining diverse functional activity of these genes across sugarcane tissues and genotypes. Sixty-six OGs were differentially expressed under stress conditions, especially cold and osmotic stresses. Gene co-expression network and functional enrichment analyses suggested that sugarcane OGs may be involved in several biological mechanisms, including stimulus response and defence mechanisms. These findings provide a valuable genomic resource for sugarcane researchers, especially those interested in selecting stress-responsive genes.


title: In-situ energy budget of needle-leaves reveals shift from evaporative to 'air cooling' under drought

abstract:
O_LIThe modulation of the leaf energy budget and the balance between its sensible heat (H) and latent heat (LE) fluxes is vital for vegetation functioning and survival, as it is linked to maintaining leaf temperature below the thermal threshold, an increasingly important mechanism under a drying and warming climate, when evaporative cooling is suppressed.
C_LIO_LICombining measurements and theoretical estimates using a new methodology, we obtained rare and comprehensive energy budgets of leaves on twigs under field conditions in droughted and non-droughted plots of a semi-arid pine forest with low and high evapotranspiration rates, respectively.
C_LIO_LIAn examination of all components of the needle-leaf energy budget indicated that under the same radiative load, leaf cooling shifts from nearly equal contributions to H and LE in non-droughted trees to almost exclusively H in droughted ones while maintaining a similar leaf temperature.
C_LIO_LIThis LE-to-H shift in leaves of droughted trees highlights the efficiency of the  air cooling mechanism in maintaining temperature, which can enhance the resilience of trees to drying conditions. Additionally, leaf energy budgets are a fundamental tool to help understand leaf cooling and aerodynamic resistance under field conditions, and to improve modelling of ecosystem activity and its effect on the climate system.
C_LI


title: A transcription factor module regulating stomatal formation is co-opted for the development of setae in the astomatous liverwort Marchantia polymorpha

abstract: The evolution of special types of cells requires the acquisition of new gene regulatory networks controlled by transcription factors (TFs). In stomatous plants, the stomatal formation is regulated by a TF module formed by subfamilies Ia and IIIb basic helix-loop-helix TFs (Ia-IIIb bHLH); however, how this module evolved during land plant diversification remains obscure. Here, we show that, in the astomatous liverwort Marchantia polymorpha, a Ia-IIIb bHLH module regulates the development of a unique sporophyte tissue, the setae, which are found in mosses and liverworts. The sole Ia bHLH gene, MpSETA, and a IIIb bHLH gene, MpICE2, regulate cell division and/or differentiation of seta lineage cells. MpSETA can partially replace the stomatal function of Ia bHLH TFs in Arabidopsis thaliana, suggesting the common regulatory mechanism underlying setal and stomatal formation. Our findings reveal the co-option of a Ia-IIIb bHLH TF module for regulating the cell fate determination and/or cell division of distinct types of cells during the evolution of land plant.


title: A pipeline for assessing the quality of images and metadata from crowd-sourced databases.

abstract: Crowd-sourced biodiversity databases provide easy access to data and images for ecological education and research. One concern with using publicly sourced databases; however, is the quality of their images, taxonomic descriptions, and geographical metadata. To address this concern and allow researchers and educators to make informed decisions about using crowd-sourced data, I developed a suite of pipelines to evaluate taxonomic consistency, how well geo-tagging fits known distributions, and image quality of crowd-sourced biodiversity data of the order Araneae (spiders) from iNaturalist. This pipeline allows users to analyze multiple images from iNaturalist and their associated metadata; to determine the level of taxonomic identification (family, genera, species) for each occurrence; whether the taxonomy label for an image matches accepted nesting of families, genera, and species; and whether geo-tags match the distribution of the taxon described using occurrence data from the Global Biodiversity Infrastructure Facility (GBIF) as a reference. Additionally, I assessed image quality with the MatLab algorithm, BRISQUE. I used entries from the order Araneae (spiders) as a case study. At the time of my analyses (July 2021), I found that iNaturalist contained at least one observation for 124 of the 129 families of Araneae, and 115 families had three or more unique observations, with relatively similar quality of metadata and image quality across families. Taxonomic consistency was similar for observations identified at the genus and species level, but lower in observations with only family level identification. Observations with species level identifications had higher precision for geo-tags compared to those identified to the family or genus level and the highest image quality according to the BRISQUE scores. Overall, the results suggest that iNaturalist can provide large metadata and images sets for research. Given the inevitability of some low-quality observations, this pipeline provides a valuable resource for researchers and educators to evaluate the quality of iNaturalist and other crowd-sourced data.

category: zoology


title: Concussive head trauma perturbs axon initial segment function in axotomized and intact layer 5 pyramidal neurons

abstract: The axon initial segment (AIS) is a critical locus of control of action potential (AP) generation and neuronal information synthesis. Concussive traumatic brain injury gives rise to diffuse axotomy, and the majority of neocortical axonal injury arises at the AIS. Consequently, concussive traumatic brain injury might profoundly disrupt the functional specialization of this region. To investigate this hypothesis, one and two days after mild central fluid percussion injury in Thy1-YFP-H mice, we recorded high-resolution APs from axotomized and adjacent intact layer 5 pyramidal neurons and applied a second derivative (2{degrees}) analysis to measure the AIS- and soma-regional contributions to the AP upstroke. All layer 5 pyramidal neurons recorded from sham animals manifested two stark 2{degrees} peaks separated by a negative intervening slope. In contrast, within injured mice, we discovered a subset of axotomized layer 5 pyramidal neurons in which the AIS-regional 2{degrees} peak was abolished, a functional perturbation associated with diminished excitability, axonal sprouting and distention of the AIS as assessed by staining for ankyrin-G. Our analysis revealed an additional subpopulation of both axotomized and intact layer 5 pyramidal neurons that manifested a melding together of the AIS- and soma-regional 2{degrees} peaks, suggesting a more subtle aberration of sodium channel function and/or translocation of the AIS initiation zone closer to the soma. When these experiments were repeated in animals in which cyclophilin-D was knocked out, these effects were ameliorated, suggesting that trauma-induced AIS functional perturbation is associated with mitochondrial calcium dysregulation.

category: neuroscience