title: A role for the circadian transcription factor NPAS2 in the progressive loss of non-rapid eye movement sleep and increased arousal during fentanyl withdrawal in male mice

abstract:
AbstractO_ST_ABSRationaleC_ST_ABSSynthetic opioids like fentanyl are contributing to the rise in rates of opioid use disorder and drug overdose deaths. Sleep dysfunction and circadian rhythm disruption may worsen during opioid withdrawal and persist during abstinence. Severe and persistent sleep and circadian alterations are putative factors in opioid craving and relapse. However, very little is known about the impact of fentanyl on sleep architecture and sleep-wake cycles, particularly opioid withdrawal. Further, circadian rhythms regulate sleep-wake cycles, and the circadian transcription factor, neuronal PAS domain 2 (NPAS2) is involved in the modulation of sleep architecture and drug reward. Here, we investigate the role of NPAS2 in fentanyl-induced sleep alterations.

ObjectivesTo determine the effect of fentanyl administration and withdrawal on sleep architecture, and the role of NPAS2 as a factor in fentanyl-induced sleep changes.

MethodsElectroencephalography (EEG) and electromyography (EMG) was used to measure non-rapid eye movement sleep (NREMS) and rapid eye movement sleep (REMS) at baseline and following acute and chronic fentanyl administration in wild-type and NPAS2-deficient male mice.

ResultsAcute and chronic administration of fentanyl led to increased wake and arousal in both wild-type and NPAS2-deficient mice, an effect that was more pronounced in NPAS2-deficient mice. Chronic fentanyl administration led to decreased NREMS, which persisted during withdrawal, progressively decreasing from day 1 to 4 of withdrawal. The impact of fentanyl on NREMS and arousal was more pronounced in NPAS2-deficient mice.

ConclusionsChronic fentanyl disrupts NREMS, leading to a progressive loss of NREMS during subsequent days of withdrawal. Loss of NPAS2 exacerbates the impact of fentanyl on sleep and wake, revealing a potential role for the circadian transcription factor in opioid-induced sleep changes.


title: Coupling of NOD2 to GIV is Required for Bacterial Sensing

abstract:
Sensing of pathogens by Nucleotide oligomerization domain (NOD)-like 2 receptor (NOD2) induces a protective inflammatory response that coordinates bacterial clearance. Polymorphisms in NOD2 impair bacterial clearance, leading to chronic gut inflammation in Crohns disease (CD) via mechanisms that remain incompletely understood. We identify GIV/Girdin (CCDC88A) as a NOD2-interactor that shapes bacterial sensing-and-signaling in macrophages. Myeloid-specific GIV depletion exacerbated and protracted infectious colitis and abolished the protective effect of muramyl dipeptide (MDP) in both chemical colitis and severe sepsis. In the presence of GIV, macrophages enhance anti-bacterial pathways downstream of NOD2, clear microbes rapidly and concomitantly suppress inflammation. GIVs actions are mediated via its C-terminus, which directly binds the terminal leucine-rich repeat (LRR#10) of NOD2; binding is augmented by MDP and ATP, precedes receptor oligomerization, and is abolished by the 1007fs CD-risk variant which lacks LRR#10. Findings illuminate mechanisms that underlie protective NOD2 signaling and loss of function in the major 1007fs variant.

GRAPHIC ABSTRACT

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/489574v1_ufig1.gif" ALT="Figure 1">
View larger version (67K):
org.highwire.dtl.DTLVardef@d8bf25org.highwire.dtl.DTLVardef@1ed2b16org.highwire.dtl.DTLVardef@1a7c8e8org.highwire.dtl.DTLVardef@770ec2_HPS_FORMAT_FIGEXP  M_FIG C_FIG In briefThis work reveals a mechanism by which macrophages use their innate immune sensor, NOD2, to protect the host against overzealous inflammation during bacterial infections, and the consequences of its loss, as occurs in the most important Crohns disease-risk variant.

HIGHLIGHTSO_LIGIV is a functional and direct interactor of the terminal LRR repeat of NOD2
C_LIO_LIMice lacking M[FE] GIV develop dysbiosis, protracted ileocolitis and sepsis
C_LIO_LIMDP/NOD2-dependent protective host responses require GIV
C_LIO_LICD-risk NOD2 1007fs variant lacking the terminal LRR#10 cannot bind GIV
C_LI

category: immunology


title: Analysis of Aedes aegypti microRNAs in response to Wolbachia wAlbB infection and their potential role in mosquito longevity

abstract:
The mosquito Aedes aegypti is the primary vector of a range of medically important viruses including dengue, Zika, West Nile, yellow fever, and chikungunya viruses. The endosymbiotic bacterium Wolbachia pipientis wAlbB strain has emerged as a promising biocontrol agent for blocking viral transmission by Ae. aegypti. In order to predict and manage the long-term efficacy of its application in the field, a thorough understanding of the molecular interactions between symbiont, host, and pathogen is required. Wolbachia are known to influence host physiology in a variety of ways including reproduction, innate immunity, metabolism, and longevity. MicroRNAs (miRNAs) are highly conserved small non-coding RNAs that are important regulators of gene expression in eukaryotes and viruses. A number of miRNAs have been functionally described in Drosophila and mosquitoes as having roles in regulating a broad range of biological processes, as well as facilitating Wolbachia maintenance. We generated the first chromosomal map of Ae. aegypti miRNAs, and compared the miRNA expression profiles between a wAlbB-transinfected Ae. aegypti mosquito line and a tetracycline cleared derivative using deep small RNA-sequencing. We found a number of miRNAs to be modulated by wAlbB infection. We also found that some miRNAs were upregulated with age in a manner that was affected by wAlbB. By selectively inhibiting some differentially expressed miRNAs, we identified miR-2946-3p and miR-317-3p as effecting mosquito longevity.

ImportanceWolbachia is an endosymbiotic bacterium found in about 65% of insect species. It is mostly known for reproductive manipulations of the host, and also blocking replication of negative sense RNA viruses. Transinfection of Wolbachia into Aedes aegypti mosquitoes, which transmit a variety of arboviruses, including dengue virus, has provided a novel biological approach in reducing transmission of arboviruses. To gain a better understanding of Wolbachia-mosquito interactions, we investigated the impact of Wolbachia on the microRNA profile of Ae. aegypti mosquitoes. Besides identifying several differentially expressed microRNAs and their putative target genes, we found alterations of microRNAs in mosquitoes due to age. Inhibition of two miRNAs led to reduced mosquito life span. The outcomes provide insights into underlying molecular mechanisms involved in Wolbachia-host interactions.

category: microbiology


title: Extensive diversity of RNA viruses in ticks revealed by metagenomics in northeastern China

abstract: Recently, several emerging tick-borne viruses have been identified to be associated with human diseases in northeastern China. Here, we used metagenomics to investigate the virome diversity in Haemaphysalis japonica, H. conicinna, Dermacentor silvarum, and Ixodes persulcatus ticks in northeastern China. A total of 22 RNA viruses were identified and belonged to more eight viral families, including four each in Nairoviridae and Phenuiviridae, three each in Flaviviridae, Rhabdoviridae, and Solemoviridae, two in Chuviridae, and one each in Partitiviridae, Tombusviridae, and unclassified. Of them, eight viruses were novel species, belonging to Nairoviridae (Jian nairovirus and Yichun nairovirus), Phenuiviridae (Mudanjiang phlebovirus), Rhabdoviridae (Tahe rhabdovirus 1-3), Chuviridae (Yichun mivirus), and Tombusviridae (Yichun tombus-like virus), and five members were established human pathogens, including Alongshan virus, tick-borne encephalitis virus, Songling virus, Beiji nairovirus, and Nuomin virus. I. persulcatus ticks had significant higher viral species than those in H. japonica, H. concinna, and D. silvarum ticks. Significant differences in tick viromes were observed among Daxingan, Xiaoxingan and Changbai mountains. These findings showed an extensive diversity of RNA viruses in ticks in northeastern China, revealed potential public health threats from the emerging tick-borne viruses. Further studies are needed to explain the natural circulation and pathogenicity of these viruses.


title: P681 mutations within the polybasic motif of spike dictate fusogenicity and syncytia formation of SARS CoV-2 variants

abstract: The rapid spread and dominance of the Omicron SARS-CoV-2 over its Delta variant has posed severe global challenges. While extensive research on the role of the Receptor Binding Domain on viral infectivity and vaccine sensitivity has been documented, the role of the spike 681PRRAR/SV687 polybasic motif is less clear. Here we monitored infectivity and vaccine sensitivity of Omicron SARS-CoV-2 pseudovirus against sera samples that were drawn four months post administration of the third dose of BNT162b2 mRNA vaccine. Our findings show that relative to Wuhan-Hu and Delta SARS-CoV-2, Omicron displayed enhanced infectivity and a sharp decline in its sensitivity to vaccine-induced neutralizing antibodies. Furthermore, while the spike proteins form Wuhan-Hu (P681), Omicron (H681) and BA.2 (H681) pseudoviruses modestly promoted cell fusion and syncytia formation, Delta spike (P681R) displayed enhanced fusogenic activity and syncytia formation capability. Live-viruses plaque formation assays confirmed these findings and demonstrated that relatively to the Wuhan-Hu and Omicron SARS-CoV-2, Delta formed more plaques that were smaller in size. Introducing a single P681R point mutation within the Wuhan-Hu spike, or H681R within Omicron spike, restored fusion potential to similar levels observed for Delta spike. Conversely, a R681P point mutation within Delta spike efficiency abolished fusion potential. We conclude that over time, the efficiency of the third dose of the Pfizer vaccine against SARS CoV-2 is waned, and cannot neutralize Omicron. We further verify that the P681 position of the viral spike dictates fusogenicity and syncytia formation.


title: The conserved histone chaperone Spt6 facilitates DNA replication and mediates genome instability

abstract: Histone chaperones are an important class of proteins that regulate chromatin accessibility for DNA-templated processes. Spt6 is a conserved histone chaperone and key regulator of transcription and chromatin structure. However, its functions outside of these roles have been little explored. In this work, we demonstrate a role for S. cerevisiae Spt6 in DNA replication and more broadly as a regulator of genome stability. Spt6 binds the replication machinery and depletion or mutation of Spt6 impairs DNA replication in vivo. Additionally, spt6 mutants are sensitive to DNA replication stress inducing agents, with increased sensitivity when combined with loss of DNA replication associated factors. Furthermore, spt6 mutants have elevated levels of DNA double strand breaks and recombination. These effects appear to be independent of R-loops, which are not elevated in spt6 mutants. Our results identify Spt6 as a regulator of genome stability, at least in part through a role in DNA replication.

category: molecular biology


title: Molecular mechanism of Afadin substrate recruitment to the receptor phosphatase PTPRK via its pseudophosphatase domain

abstract: Protein tyrosine phosphatase receptor-type kappa (PTPRK) is a transmembrane receptor that links extracellular homophilic interactions to intracellular catalytic activity. Previously we showed that PTPRK promotes cell-cell adhesion by selectively dephosphorylating several cell junction regulators including the protein Afadin (Fearnley et al., 2019). Here we demonstrate that Afadin is recruited for dephosphorylation by directly binding to the PTPRK D2 pseudophosphatase domain. We mapped this interaction to a putative coiled coil (CC) domain in Afadin that is separated by more than 100 amino acids from the substrate pTyr residue. We identify the residues that define PTP specificity, explaining how Afadin is selectively dephosphorylated by PTPRK yet not by the closely-related receptor tyrosine phosphatase PTPRM. Our work demonstrates that PTP substrate specificity can be determined by protein-protein interactions distal to the active site. This explains how PTPRK and other PTPs achieve substrate specificity despite a lack of specific sequence context at the substrate pTyr. Furthermore, by demonstrating that these interactions are phosphorylation-independent and mediated via binding to a non-catalytic domain, we highlight how receptor PTPs could function as intracellular scaffolds in addition to catalyzing protein dephosphorylation.


title: Mediator-RNA Polymerase II Interactions Critical for Transcriptional Activation Are Mediated by the N-terminal Half of MED14 and C-terminal Domain of RPB1

abstract: Mediator is a large and evolutionarily conserved coactivator complex essential for RNA polymerase II (Pol II)-mediated gene regulation at multiple steps of the transcription process, including preinitiation complex (PIC) assembly and function. Here, we used the MultiBac baculovirus expression system to generate recombinant human core Mediator subcomplexes and subsequent biochemical approaches to dissect the mechanism by which Mediator facilitates direct recruitment of Pol II to core promoters. Our results highlight a pivotal role in this process for the N-terminal half (NTD) of the MED14 subunit. We show that a reconstituted 15-subunit human core Mediator complex that contains only the MED14-NTD is fully functional in facilitating both basal and activated (p53) transcription. This complex directly interacts with the C-terminal domain (CTD) of the RPB1 subunit of Pol II (RPB1 CTD) and is required for recruiting Pol II to core promoters. Moreover, recombinant RPB1 can completely reverse the human core Mediator-Pol II interaction. Notably, the human MED14-NTD region has secondary structure conservation with Schizosaccharomyces pombe. In addition, reanalysis of published cryo-EM structures of yeast Mediator-Pol II complexes strongly supports our conclusion. Thus, our analyses provide critical new insights into how Mediator binds to Pol II and recruits it to the promoters to facilitate transcription.


title: Functional genomics analysis identifies impairment of HNF1B function as a cause of Mayer-Rokitansky-Küster-Hauser syndrome.

abstract: Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome is a congenital condition characterized by aplasia or hypoplasia of the uterus and vagina in women with a typical 46,XX karyotype. This condition can occur as type I when isolated or as type II when associated with extragenital anomalies including kidney and skeletal abnormalities. The genetic basis of MRKH syndrome remains unexplained and several candidate genes have been proposed to play a role in its etiology, including HNF1B, LHX1, and WNT4. Here, we conducted a genomic analysis of 13 women affected by MRKH syndrome, resulting in the identification of candidate genes, including several novel candidates. We focused on HNF1B for further investigation due to its known association with, but unknown etiological role in, MRKH syndrome. We ablated Hnf1b specifically in the epithelium of the Mullerian ducts in mice, and found that this caused hypoplastic development of both the epithelial and stromal compartments of the uterus, as well as kidney anomalies, closely mirroring the MRKH type II phenotype. Using single-cell RNA sequencing of uterine tissue in the Hnf1b-ablated embryos, we analyzed the molecules and pathways downstream of Hnf1b, revealing a dysregulation of processes associated with cell proliferation, migration, and differentiation. Thus, we establish that loss of Hnf1b function leads to an MRKH phenotype, and generate the first mouse model of MRKH syndrome type II. Our results support the diagnostic value of HNF1B in clinical genetic testing for MRKH syndrome, and shed new light on the genetic causes of this poorly understood condition in womens reproductive health.

category: developmental biology


title: CLASP1 is essential for neonatal lung function and survival in mice

abstract: Life-threatening breathing problems due to incomplete lung development or acute respiratory distress are common in premature babies and respiratory diseases. Here we show that the microtubule plus-end-tracking protein CLASP1 is required for neonatal lung function and survival. CLASP1 is expressed in the lungs and associated respiratory structures throughout embryonic development. Clasp1 disruption in mice caused intrauterine growth restriction and neonatal lethality due to acute respiratory failure. Knockout animals showed impaired lung inflation associated with smaller rib cage formation, delayed fetal lung maturation and diaphragms with abnormal innervation. Live-cell analysis of microtubule dynamics in cultured hippocampal neurons revealed an increased catastrophe rate, consistent with a role of CLASP1 in neurite outgrowth. Gene expression studies further indicated that CLASP1 is required for normal lung cell differentiation. These findings unveil an unexpected link between the regulation of microtubule dynamics, respiratory muscle innervation and lung cell differentiation, essential for neonatal lung function and survival.


title: Mutation rates and adaptive variation among the clinically dominant clusters of Mycobacterium abscessus

abstract: Mycobacterium abscessus (Mab) is a multi-drug resistant pathogen increasingly responsible for severe pulmonary infections. Analysis of whole genome sequences (WGS) of Mab demonstrates dense genetic clustering of clinical isolates collected from disparate geographic locations. This has been interpreted as supporting patient-to-patient transmission, but epidemiological studies have contradicted this interpretation. Here we present evidence for a slowing of the Mab molecular clock rate coincident with the emergence of phylogenetic clusters. We find that clustered isolates are enriched in mutations affecting DNA repair machinery and have lower spontaneous mutation rates in in vitro. We propose that Mab adaptation to the host environment through variation in DNA repair genes affects the organisms mutation rate and that this manifests as phylogenetic clustering. These results inform our understanding of niche switching for facultative pathogens, and challenge the model of transmission as the major mode of dissemination of clinically dominant Mab clusters.

category: genomics


title: Cocoa Extract Exerts Sex-Specific Effects in an Aggressive Hyperglycemia Model: A Pilot Study

abstract: Type 2 diabetes (T2D) is characterized by hyperglycemia and insulin resistance. Cocoa may slow T2D development and progression. This study employed male and female BTBR.Cg-Lepob/ob/WiscJ and wild type (WT) controls to assess the potential for cocoa to ameliorate progressive T2D and compare responses between sexes. Mice received diet without (WT, ob/ob) or with cocoa extract (ob/ob + c) for 10 weeks. Glucose and insulin tolerance tests (GTT/ITT) were conducted at weeks 1, 5 and 2, 6, respectively. Cocoa provided mild non-significant protection against weight gain vs. ob/ob control in males but not females. Male ob/ob + c had increasing fasting glucose at weeks 1 and 5 GTTs, with significantly higher levels of fasting glucose than ob/ob control at week 5. This was not seen in females. Cocoa protected against elevated 4-hour fasting glucose in week 2, but not week 6, ITTs. Cocoa partly suppressed hyperinsulinemia in males but significantly amplified it in females and protected against loss of beta cell area in females only. The mechanisms of these sex-specific effects remain to be elucidated. This study informs additional experiments with larger sample sizes and demonstrates that sex differences must be considered when designing dietary interventions for T2D.

category: biochemistry


title: Inhibiting succinate release worsens cardiac reperfusion injury by enhancing mitochondrial reactive oxygen species generation

abstract: The metabolite succinate accumulates during cardiac ischemia. Within 5 min. of reperfusion, succinate returns to baseline levels via both its release from cells and oxidation by mitochondrial complex II (Cx-II). The latter drives reactive oxygen species (ROS) generation and subsequent opening of the mitochondrial permeability transition (PT) pore, leading to cell death. Targeting succinate dynamics (accumulation/oxidation/release) may be therapeutically beneficial in cardiac ischemia-reperfusion (IR) injury. It has been proposed that blocking monocarboxylate transporter 1 (MCT-1) may be beneficial in IR, by preventing succinate release and subsequent engagement of downstream inflammatory signaling pathways. In contrast, herein we hypothesized that blocking MCT-1 would retain succinate in cells, exacerbating ROS generation and IR injury. Using the mitochondrial ROS probe mitoSOX, and a custom-built murine heart perfusion rig built into a spectrofluorometer, we measured ROS generation in-situ during the first moments of reperfusion, and found that acute MCT-1 inhibition enhanced mitochondrial ROS generation at reperfusion, and worsened IR injury (recovery of function and infarct size). Both these effects were abrogated by tandem inhibition of Cx-II, suggesting that succinate retention worsens IR due to driving more mitochondrial ROS generation. Furthermore, using the PT pore inhibitor cyclosporin A, along with monitoring of PT pore opening via the mitochondrial membrane potential indicator TMRE, we herein provide evidence that ROS generation during early reperfusion is upstream of the PT pore, not downstream as proposed by others. In addition, pore opening was exacerbated by MCT-1 inhibition. Together, these findings highlight the importance of succinate dynamics and mitochondrial ROS generation, as key determinants of PT pore opening and IR injury outcomes.


title: Computationally Guided Design of Novel Angiotensin Converting Enzyme Inhibitors

abstract: Angiotensin-converting enzyme inhibitors (ACEI) such as Moexipril, Trandolapril, Ramipril, and Perindopril are used to treat hypertension. However, these drugs have undesired side effects. Using computational studies, two new novel drug candidates were designed with improved molecular interactions, ADMET properties, and docking scores compared to Moexipril. Homology analysis was done to determine the best animal model for preclinical studies, and it revealed that Pan troglodytes (chim-panzees) and Mus musculus (house mouse) were the best candidates.


title: A conserved signaling pathway activates bacterial CBASS immune signaling in response to DNA damage

abstract: To protect themselves from the constant threat of bacteriophage (phage) infection, bacteria have evolved diverse immune systems including restriction/modification, CRISPR/Cas, and many others. Here we describe the discovery of a two-protein transcriptional regulator module associated with hundreds of CBASS (Cyclic oligonucleotide Based Anti-phage Signaling System) immune systems, and demonstrate that this module drives expression of its associated CBASS system in response to DNA damage. We show that the helix-turn-helix transcriptional repressor CapH binds the promoter region of its associated CBASS system to repress transcription until it is cleaved by the metallopeptidase CapP. CapP is inactive except in the presence of single-stranded DNA, and CapP activity in cells is stimulated by DNA-damaging drugs. Together, CapH and CapP drive increased expression of their associated CBASS system in response to DNA damage. In both their structures and mechanisms, CapH and CapP resemble regulators that drive increased expression of DNA damage response genes in radiation-resistant Deinococcus, and control the mobilization of prophages and mobile elements in response to DNA damage. We also identify CapH and CapP-related proteins associated with diverse known and putative bacterial immune systems, including DISARM and two uncharacterized operons encoding proteins related to eukaryotic ubiquitin signaling pathways. Overall, our data highlight a mechanism by which bacterial immune systems can sense and respond to a universal stress signal, potentially enabling multiple immune systems to mount a coordinated defensive effort against an invading pathogen.


title: Pericytes and Astrocytes Instruct Glioblastoma Invasion, Proliferation, and Therapeutic Response within an Engineered Brain Perivascular Niche Model

abstract: Glioblastoma (GBM) tumor cells are found in the perivascular niche microenvironment and are believed to associate closely with the brain microvasculature. However, it is largely unknown how the resident cells of the perivascular niche, such as endothelial cells, pericytes, and astrocytes, influence GBM tumor cell behavior and disease progression. We describe a three-dimensional in vitro model of the brain perivascular niche developed by encapsulating brain-derived endothelial cells, pericytes, and astrocytes in a gelatin hydrogel. We show that pericytes and astrocytes explicitly contribute to vascular architecture and maturation. We use co-cultures of patient-derived GBM tumor cells with brain microvascular cells to identify a role for pericytes and astrocytes in establishing a perivascular niche environment that modulates GBM cell invasion, proliferation, and therapeutic response. Engineered models provides unique insight regarding the spatial patterning of GBM cell phenotypes in response to a multicellular model of the perivascular niche. Critically, we show that engineered perivascular models provide an important resource to evaluate mechanisms by which inter- cellular interactions modulate GBM tumor cell behavior, drug response, and provide a framework to consider patient-specific disease phenotypes.

category: bioengineering


title: Biomolecular condensates govern PARP inhibitor trapping and present mechanisms of resistance

abstract: Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) are a class of cancer drugs that enzymatically inhibit PARP activity at sites of DNA damage. In the context of BRCA mutations, PARPi can be synthetically lethal, presenting ideal genetic targeting. Yet, PARPi function primarily by trapping PARP1 onto sites of DNA damage. How PARPi trap and why some are better trappers remain unknown. Here, we show trapping occurs primarily through a kinetic phenomenon within biomolecular condensates that correlates with PARPi koff. Our results suggest PARP trapping is not the physical stalling of PARP1 on DNA, rather the high probability of PARP re-binding damaged DNA in the absence of other DNA binding protein recruitment. Furthermore, we found recruitment of the DNA binding protein RPA1 correlates to cell line PARPi sensitivity independent of RPA1 expression, demonstrating that condensate recruitment alone can impact efficacy. These results shed new light on how PARPi function, describe how PARPi properties correlate to trapping potency, and suggest previously unknown mechanisms of PARPi resistance.

category: biophysics


title: Loss of Myosin-1e biases MMTV-PyMT induced breast cancer towards a 4 differentiated and secretory state

abstract: Expression of the unconventional myosin, Myosin-1e (Myo1e), has been shown to contribute to tumor progression in the MMTV-PyMT mouse model of mammary tumorigenesis and is associated with poor outcome in breast cancer patients. However, the specific effects of Myo1e expression on the mammary tumor cells remain unidentified. Here, we used Myo1e-KO and wild-type (WT) MMTV-PyMT mice on a pure genetic background to further investigate the molecular and cellular effects of Myo1e expression. Myo1e-WT tumors were characterized by an increased abundance of intra-epithelial macrophages and lower amounts of the extracellular matrix. Transcriptomic profiling of WT and Myo1e-KO tumors identified a pattern of differential expression of tumor suppressor and tumor-promoting genes that was consistent with the observed differences in tumor progression and morphology between the genotypes, and also revealed differential expression of genes associated with secretion and cell-cell adhesion. In agreement with the RNA-seq findings, Myo1e-expressing tumor cells exhibited increased proliferation and elevated nuclear enrichment of YAP1 transcriptional activator compared to Myo1e-KO tumor cells. To investigate tumor cell-autonomous effects of Myo1e expression, we used the epithelial cell line PY-230 derived from the MMTV-PyMT-induced mouse tumor to create Myo1e-depleted cells by Crispr-mediated genome editing. Cells deficient in Myo1e had increased expression of genes encoding milk components compared to the wild-type cells. Electric cell-substrate impedance sensing (ECIS) measurements showed that depletion of Myo1e in PY-230 cells resulted in increased resistance to electrical current indicating enhanced epithelial barrier function. Overall, we find that Myo1e expression biases tumors towards a less-differentiated, pro-tumorigenic state, and that depletion of Myo1e is associated with a pro-secretory, more differentiated state.

category: cancer biology


title: Fic-mediated AMPylation tempers the Unfolded Protein Response during physiological stress

abstract: The proper balance of synthesis, folding, modification and degradation of proteins, also known as protein homeostasis, is vital to cellular health and function. The unfolded protein response (UPR) is activated when the mechanisms maintaining protein homeostasis in the endoplasmic reticulum (ER) become overwhelmed. However, prolonged or strong UPR responses can result in elevated inflammation and cellular damage. Previously, we discovered that the bifunctional enzyme Fic can modulate the UPR response via post-translational modification of BiP by AMPylation and deAMPylation. Loss of fic in Drosophila leads to vision defects and altered UPR activation in the fly eye. To investigate the importance of Fic-mediated AMPylation in a mammalian system, we generated a conditional null allele of Fic in mice and characterized the effect of Fic loss on the exocrine pancreas. Compared to controls, Fic-/- mice exhibit elevated serum markers for pancreatic dysfunction and display enhanced UPR signaling in the exocrine pancreas in response to physiologic and pharmacological stress. In addition, both fic-/- flies and Fic-/- mice show reduced capacity to recover from damage by stress that triggers the UPR. These findings show that Fic- mediated AMPylation acts as a molecular rheostat that is required to temper the UPR response in the mammalian pancreas during physiological stress.

category: physiology


title: Integrative human atrial modeling unravels interactive PKA and CaMKII signaling as key determinant of atrial arrhythmogenesis

abstract:
Atrial fibrillation (AF), the most prevalent clinical arrhythmia, is associated with atrial remodeling manifesting as acute and chronic alterations in expression, function, and regulation of atrial electrophysiological and Ca2+-handling processes. These AF-induced modifications crosstalk and propagate across spatial scales creating a complex pathophysiological network, which renders AF resistant to existing pharmacotherapies that predominantly target transmembrane ion channels. Developing innovative therapeutic strategies requires a systems approach to disentangle quantitatively the proarrhythmic contributions of individual AF-induced alterations. Here, we built a novel computational framework for simulating electrophysiology and Ca2+-handling in human atrial cardiomyocytes and tissues, and their regulation by key upstream signaling pathways (i.e., protein kinase A, PKA, and Ca2+/calmodulin-dependent protein kinase II, CaMKII) involved in AF-pathogenesis. Populations of atrial cardiomyocyte models were constructed to determine the influence of subcellular ionic processes, signaling components, and regulatory networks on atrial arrhythmogenesis. Our results reveal a novel synergistic crosstalk between PKA and CaMKII that promotes atrial cardiomyocyte electrical instability and arrhythmogenic triggered activity. Simulations of heterogeneous tissue demonstrate that this cellular triggered activity is further amplified by CaMKII-dependent alterations of tissue properties, further exacerbating atrial arrhythmogenesis. Our analysis positions CaMKII as a key nodal master switch of the adaptive changes and the maladaptive proarrhythmic triggers at the cellular and tissue levels and establishes CaMKII inhibition as potential anti-AF strategy. Collectively, our integrative approach is powerful and instrumental to assemble and reconcile existing knowledge into a systems network for identifying novel anti-AF targets and innovative approaches moving beyond the traditional ion channel-based strategy.

Significance statementDespite significant advancement in our understanding of pathological mechanisms and alterations underlying atrial fibrillation (AF), a highly prevalent clinical arrhythmia causing substantial health and socioeconomic burden, development of effective pharmacological therapeutics for AF remains an urgent unmet clinical need. We built a systems framework integrating key processes and their regulatory upstream signaling pathways that are involved in atrial electrophysiology and modified by AF. By simulating populations of single atrial cardiomyocyte models and heterogeneous tissues, our analysis demonstrated synergistic interactions between upstream signaling pathways that promote atrial arrhythmogenesis across spatial scales, added new insight into complex atrial arrhythmia mechanisms, and revealed adaptive and maladaptive alterations caused by AF, thus providing a powerful new tool for identifying innovative therapeutic approaches against AF.


title: T32 training is associated with increased likelihood of obtaining an academic research faculty position: a cross-sectional study

abstract:
BackgroundThe main mission of NIH-sponsored institutional training programs such as the T32 is to provide strong research and career training for early career scientists, while preparing those individuals to become leaders working to meet the health-related research needs of the nation. One of the main avenues to pursue health-related research is becoming research faculty at an academic institution. It is therefore important to know whether these programs are succeeding in this mission, or, if barriers exist that prevent trainees from pursuing these careers.

MethodsOur institution currently trains ~ 2400 post-doctoral scholars per year, approximately 5% of whom are enrolled in one of our 33 T32 programs. In this study, we 1) compare the professional outcomes of T32 trainees with non-T32 trainees at our institution, and 2) survey past and current T32 trainees in a subset of high-performing cardiovascular programs about the barriers and enablers they experienced to pursuing research-oriented careers.

ResultsFormer T32 trainees are significantly more likely to attain appointments as primarily research faculty members, compared to other trainees. Trainees report a perceived lack of stability, the paucity of open positions, and the  publish or perish competitive mentality of academia as their top reasons for abandoning careers in academia. However, they were still more likely to choose research over clinical careers after participating in a dedicated T32 program.

ConclusionsOur results support the conclusion that structured training programs strengthen the pipeline of young scientists pursuing careers in academic research, including those from underrepresented backgrounds. However, T32 postdoctoral researchers are held back from pursuing academic careers by a perceived lack of stability and high competition for faculty positions.

FundingThis research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

category: scientific communication and education


title: Computational analysis of 4-1BB-induced NF	κB signaling suggests improvements to CAR cell design

abstract:
BackgroundChimeric antigen receptor (CAR)-expressing cells are a powerful modality of adoptive cell therapy against cancer. The strength and dynamics of signaling events initiated upon antigen binding depend on the costimulatory domain within the structure of the CAR. One such costimulatory domain is 4-1BB, which affects cellular response via the NF{kappa}B pathway. However, the quantitative aspects of 4-1BB-induced NF{kappa}B signaling are not fully understood.

MethodsWe developed an ordinary differential equation-based mathematical model representing canonical NF{kappa}B signaling activated by CAR-4-1BB. We first performed a global sensitivity analysis on our model to quantify the impact of kinetic parameters and initial protein concentrations. We ran Monte Carlo simulations of cell population-wide variability in NF{kappa}B signaling and used Kraskovs algorithm to quantify the mutual information between the extracellular signal and different levels of the NF{kappa}B signal transduction pathway.

ResultsWe determined that in response to a wide range of antigen concentrations, the magnitude of the transient peak in the nuclear concentration of NF{kappa}B varies significantly, while the timing of this peak is relatively consistent. Our global sensitivity analysis showed that the model is robust to variations in parameter values, and thus, its quantitative predictions would remain applicable to a broad range of parameter values. Next, we found that overexpressing NEMO and disabling IKK{beta} deactivation are predicted to increase the mutual information between antigen levels and NF{kappa}B activation.

ConclusionsOur modeling predictions provide actionable insights to guide CAR development. Particularly, we propose specific manipulations to the NF{kappa}B signal transduction pathway that can fine-tune the response of CAR-4-1BB cells to the antigen concentrations they are likely to encounter.

category: systems biology


title: Resource allocation accounts for the large variabilityof rate-yield phenotypes across bacterial strains

abstract: Different strains of a microorganism growing in the same environment display a wide variety of growth rates and growth yields. We developed a coarse-grained model to test the hypothesis that different resource allocation strategies, corresponding to different compositions of the proteome, can account for the observed rate-yield variability. The model predictions were verified by means of a database of hundreds of published rate-yield and flux phenotypes of Escherichia coli strains grown in standard laboratory conditions. We found a very good quantitative agreement between the predicted and observed variability in rates and yields. Moreover, over the entire range of wild-type and mutant strains considered, acetate overflow was predicted not to correlate with the growth rate, in agreement with the experimental data. These results support the hypothesis that resource allocation is a major explanatory factor of the observed variety of growth rates and growth yields across different bacterial strains. We also show, however, that differences in enzyme activity need to be taken into account to explain variations in protein abundance. Our model allows a fundamental understanding of quantitative bounds on rate and yield in E. coli and other microorganisms. It may also be useful for the rapid screening of strains in metabolic engineering and synthetic biology.


title: Humanization and Expression of IgG and IgM Antibodies in Plants as Potential Diagnostic Reagents for Valley Fever

abstract: Monoclonal antibodies (mAbs) are important proteins used in many life science applications, from diagnostics to therapeutics. High demand for mAbs for different applications urges the development of rapid and reliable recombinant production platforms. Plants provide a quick and inexpensive system for producing recombinant mAbs. Moreover, when paired with an established platform for mAb discovery, plants can easily be tailored to produce mAbs of different isotypes against the same target. Here, we demonstrate that a hybridoma-generated mouse mAb against chitinase 1 (CTS1), an antigen from Coccidioides spp., can be biologically engineered for use with serologic diagnostic test kits for coccidioidomycosis (Valley Fever) using plant expression. The original mouse IgG was modified and recombinantly produced in plants as IgG and IgM isotypes with human kappa, gamma, and mu constant regions. The two mAb isotypes produced in plants were shown to maintain target antigen recognition to CTS1 using similar reagents as the Food and Drug Administration (FDA)-approved Valley Fever diagnostic kits. As none of the currently approved kits provide antibody dilution controls, humanization of antibodies that bind to CTS1, a major component of the diagnostic antigen preparation, may provide a solution to the lack of consistently reactive antibody controls for Valley Fever diagnosis. Furthermore, our work provides a foundation for reproducible and consistent production of recombinant mAbs engineered to have a specific isotype for use in diagnostic assays.


title: Selection of software and database for metagenomics sequence analysis impacts the outcome of microbial profiling and pathogen detection

abstract:
AimShotgun metagenomic sequencing analysis is widely used for microbial profiling of biological specimens and pathogen detection. However, very little is known about the technical biases caused by the choice of analysis software and databases. In this study, we evaluated shotgun metagenomics taxonomical profiling software to characterize the microbial compositions of biological samples collected from wild rodents.

Method and ResultsUsing nine of the most widely used metagenomics software and four different databases, we analyzed shotgun metagenomic sequence data from three sets of wild rodent tissue samples. We demonstrated the discrepancies in results when different databases and software were used, which cause significant variation in microbial community characterizations. Our analysis also showed that these software differed in their ability to detect the presence of Leptospira, a major zoonotic pathogen of one health importance.

ConclusionsSignificant differences in compositional profiles for the same dataset while using different databases and software combinations can result in confounding biological conclusions in microbial profiling.

Significance and Impact of StudyThis study cautions that the selection of software and databases to analyze metagenomics data can influence the outcome and biological interpretation.


title: Generation of Gausemycin A-resistant Staphylococcus aureus

abstract:
Gausemycins A and B are the first members of the novel lipoglycopeptides family produced by Streptomyces roseoflavus INA-Ac-5812, which showed the ability to fight clinically important Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus. However, new antibiotics need to be studied in depth to determine their full potential. In this study, we concentrated our efforts to investigate resistance emerging within S. aureus upon gausemycin A application.

Using serial passaging of S. aureus FDA209P in increasing concentrations of gausemycin A, we obtained the resistant variant S. aureus 5812R which are 80-times more resistant comparing to the origin strain. Moreover, obtained resistance is stable, since 15 passages in a drug-free medium did not restore bacterial susceptibility to gausemycin A.

Elucidating of the differences between resistant and parent strains was concerned antibiotic cross-resistance, structure of bacterial membrane, and response at genetic level.

Susceptibility testing of S. aureus 5812R revealed the acquisition of cross-resistance to daptomycin, cefazolin, and tetracycline, while resistance to vancomycin, nisin and ramoplanin absence. The composition of fatty acids constituting the cytoplasmic membrane of S. aureus 5812R, was represented by increased content of anteiso- branched chain fatty acids, while iso-branched chain fatty acids was decreased comparing the origin S. aureus FDA209P strain. The relative expression of the cls gene catalyzing the synthesis of cardiolipin in the resistant cells was higher compared to the S. aureus FDA209P.


title: Diversity and abundance of archaeal amoA genes in the permanent and temporary oxygen minimum zones of Indian Ocean.

abstract: Oxygen minimum zones are results of oxygen consumption exceeding the oxygen availability in stratified water columns of the marine environment. We compared the ammonia monooxygenase subunit A (amoA) gene abundance and the diversity of ammonia-oxidising archaea (AOA) in the Arabian Sea (AS) with those of the Bay of Bengal (BoB). Three primer pairs targeting amoA genes of water column A (WCA), water column B (WCB) and total AOA (amoAt) captured different densities of gene copy numbers in both marginal seas. Water column A (WCA) ecotypes were more abundant in the AS than in the BoB. Core-OMZ depths of the BoB contained 10 times lower amoA copy numbers than those of the AS. Along with sampling depth, concentration of ammonia shapes the WCA/WCB ecotypes in AS/BoB. Among the total AOA populations, WCB ecotypes were more abundant. The amoA gene sequences were either of Nitrosopumilales or Ca. Nitrosotaleales members and belonged to NP-{gamma}, NP-{delta}, NS-{beta}, NS-{gamma} and NS-{varepsilon} sub-clades. Pairwise distance and nucleotide diversity index analysis reveals that BoB nurtures two times more diverse amoA sequences than the AS. The core OMZ region of the BoB contains a two-fold higher diversity of amoA gene sequences compared to the AS, whereas the AS contains 13 times more abundant amoA copies than the BoB.


title: Paradoxical decrease of imitation performance with age in children

abstract:
We studied the development of imitation ability in a cohort of 6-11-year-old children (N=174) with specific attention to error types and to their cognitive interpretation. Participants imitated meaningless actions as if they were in front of a mirror (specularly). Actions varied across three levels of complexity (movements of a single limb, of arm and leg of the same body side, or of arm and leg of opposite sides). Overall performance improved with age. Among the most frequent error categories, "side" errors (movement imitated with the left instead of the right limb or vice-versa) paradoxically increased with age (from 9 years), but their sensitivity to complexity decreased with age. Thus, encoding two opposite body sides has high cognitive costs in younger children, and smaller or null costs in older children. We interpreted these results in terms of enhancement of Working Memory (WM) and body knowledge with age. When WM increases, it paradoxically drives older children to apply their superior body knowledge and imitate "anatomically", producing side errors. Younger children are free from such interference because they still lack the necessary body knowledge and WM capacity. The (fewer) side errors made by younger children would be due to failure to manage the conflict between opposite spatial codes, an effect of limited WM capacity witnessed by their sensitivity to action complexity. In conclusion, this study suggests that anatomical imitation becomes available in childrens cognitive repertoire from age 9, as an effect of increased body knowledge and WM capacity.

Research HighlightsO_LIThe analysis of error types uncovers/illustrates the role of working memory and body knowledge in imitation.
C_LIO_LIAs an effect of little working memory capacity, younger children pay a cognitive cost to encode opposite body sides and make "side errors".
C_LIO_LIImitation performance improves with age, but side errors paradoxically increase in frequency due to greater ability to master body knowledge bringing to anatomical imitation.
C_LIO_LIAnatomical imitation is available in childrens cognitive repertoire from age 9, as an effect of the development of body knowledge and working memory capacity.
C_LI

category: animal behavior and cognition


title: Neural Implementation of Precise Temporal Patterns in Motor Cortex

abstract: One of the most concerned problems in neuroscience is how neurons communicate and convey information through spikes. There is abundant evidence in sensory systems to support the use of precise timing of spikes to encode information. However, it remains unknown whether precise temporal patterns could be generated to drive output in the primary motor cortex (M1), a brain area containing ample recurrent connections that may destroy temporal fidelity. Here, we used a novel brain-machine interface that mapped the temporal order and precision of motor cortex activity to the auditory cursor and reward to guide the generation of precise temporal patterns in M1. During the course of learning, rats performed the "temporal neuroprosthetics" in a goal-directed manner with increasing proficiency. Precisely timed spiking activity in M1 was volitionally and robustly produced under this "temporal neuroprosthetics", demonstrating the feasibility of M1 implementing temporal codes. Population analysis showed that the local network was coordinated in a fine time scale as the overall excitation heightened. Furthermore, we found that the directed connection between neurons assigned to directly control the output ("direct neurons") was strengthened throughout learning, as well as connections in the subnetwork that contains direct neurons. Network models revealed that excitatory gain and strengthening of subnetwork connectivity transitioned neural states to a more synchronous regime, which improved the sensitivity for coincidence detection and, thus, the precision of spike patterns. Therefore, our results suggested the recurrent connections facilitate the implementation of precise temporal patterns instead of impairing them, which provided new perspectives on the fine-timescale activity and dynamics of M1.

category: neuroscience


title: Estimation of the reference lead (Pb) concentration levels affecting immune cells in the blood of Black-headed Gulls (Chroicocephalus ridibundus, Laridae)

abstract: The biological effects of lead (Pb) contamination have been reported in various species. There are no restrictions on the use of Pb products, including bullets, in the areas south of Hokkaido, Japan. Local governments have announced the presence of some Pb in the soil sediments of water bodies. Previous studies have confirmed the relationship between blood Pb level (BLL) and immune cells. This study was performed with the aim of clarifying the effect of Pb contamination on immune cells. In total, 170 Black-headed Gulls (Chroicocephalus ridibundus) were captured, including a population in Tokyo Bay between November 2018 and April 2021 and a population in Mikawa Bay between January 2019 and April 2021. Linear regression analysis was performed with the white blood cell count (WBC), proportion of heterophils (Het), proportion of lymphocytes (Lym), ratio of heterophils and lymphocytes (H/L ratio), copy number of CD4 messenger RNA, and copy number of CD8 messenger RNA as the objective variables, and the BLL as the explanatory variable. The group with BLL < 1.0 g/dL had a significantly lower Het and higher Lym than that with BLL > 3.5 g/dL (P < 0.05). In addition, the group with BLL < 1.0 g/dL had a significantly lower H/L ratio than that with BLL > 3.5 g/dL. CD8 and WBC were higher in the group with the group with BLL range, from 1.0 to 3.5 g/dL than those in the group with BLL < 1.0 g/dL. This study suggests that the effect of Pb pollution on the immune cells of Black-headed Gulls is lower than some previous criteria values.

category: ecology


title: Molecular docking, simulation and binding free energy analysis of small molecules as PfHT1 inhibitors

abstract: Malaria chemotherapy has been plagued by parasite resistance. Novel drugs must be continually explored for malaria treatment. Plasmodium falciparum requires host glucose for survival and proliferation. Protein involved in hexose permeation, P. falciparum hexose transporter 1 (PfHT1) is a potential drug target. We performed high throughput virtual screening of 21,352 small-molecule compounds against PfHT1. The stability of the lead compound complexes was evaluated via molecular dynamics (MD) simulation for 100 nanoseconds. We also investigated the pharmacodynamic, pharmacokinetic and physiological characteristics of the compounds in accordance with Lipinksi rules for drug-likeness to bind and inhibit PfHT1. Molecular docking and free binding energy analyses were carried out using Molecular Mechanics with Generalised Born and Surface Area (MMGBSA) solvation to determine the selectivity of the hit compounds for PfHT1 over the human glucose transporter (hGLUT1) orthologue. Five important compounds were identified: Hyperoside (CID5281643); avicularin (CID5490064); sylibin (CID5213); harpagoside (CID5481542) and quercetagetin (CID5281680). The compounds formed intermolecular interaction with the binding pocket of the target via conserved amino acid residues (Val314, Gly183, Thr49, Asn52, Gly183, Ser315, Ser317, and Asn48). The MMGBSA analysis of the complexes yielded high free binding energies. Four (CID5281643, CID5490064, CID5213, and CID5481542) of the identified compounds were found to be stable within the PfHT1 binding pocket throughout the 100 nanoseconds simulation run time. The four compounds demonstrated higher affinity for PfHT1 than the human major glucose transporter (hGLUT1). This investigation demonstrates the inhibition potential of sylibin, hyperoside, harpagoside, and avicularin against PfHT1 receptor. Robust preclinical investigations are required to validate the chemotherapeutic properties of the identified compounds.

category: bioinformatics


title: Light diffusion in layered media: A numerical study in the spatial and time-domains

abstract: Accurate and efficient forward models of photon migration in heterogeneous geometries are important for many applications of light in medicine because many biological tissues exhibit a layered structure, with each layer having independent optical properties and thickness. Unfortunately, closed form analytical solutions are not readily available for layered tissue-models, and often are modeled using computationally expensive numerical techniques or theoretical approximations that limit accuracy and real-time analysis. Here, we develop an open-source accurate, efficient, and stable numerical routine to solve the diffusion equation in the steady-state and time-domain for a layered cylinder tissue model with an arbitrary number of layers and specified thickness and optical coefficients. We show that the steady-state (< 0.1 ms) and time-domain (< 0.5 ms) fluence (for an 8-layer medium) can be calculated with absolute numerical errors approaching machine precision. The numerical implementation increased computation speed by 3 to 4 orders of magnitude compared to previously reported theoretical solutions in layered media. We verify our solutions asymptotically to homogeneous tissue geometries using closed form analytical solutions to assess convergence and numerical accuracy. Approximate solutions to compute the reflected intensity are presented which can decrease the computation time by an additional 2-3 orders of magnitude. We also compare our solutions for 2, 3, and 5 layered media to gold-standard Monte Carlo simulations in layered tissue models of high interest in biomedical optics (e.g. skin/fat/muscle and brain). The presented routine could enable more robust real-time data analysis tools in heterogeneous tissues that are important in many clinical applications such as functional brain imaging and diffuse optical spectroscopy.


title: Decoding Surface Fingerprints for Protein-Ligand Interactions

abstract: AO_SCPLOWBSTRACTC_SCPLOWSmall molecules have been the preferred modality for drug development and therapeutic interventions. This molecular format presents a number of advantages, e.g. long half-lives and cell permeability, making it possible to access a wide range of therapeutic targets. However, finding small molecules that engage "hard-to-drug" protein targets specifically and potently remains an arduous process, requiring experimental screening of extensive compound libraries to identify candidate leads. The search continues with further optimization of compound leads to meet the required potency and toxicity thresholds for clinical applications. Here, we propose a new computational workflow for high-throughput fragment-based screening and binding affinity prediction where we leverage the available protein-ligand complex structures using a state-of-the-art protein surface embedding framework (dMaSIF). We developed a tool capable of finding suitable ligands and fragments for a given protein pocket solely based on protein surface descriptors, that capture chemical and geometric features of the target pocket. The identified fragments can be further combined into novel ligands. Using the structural data, our ligand discovery pipeline learns the signatures of interactions between surface patches and small pharmacophores. On a query target pocket, the algorithm matches known target pockets and returns either potential ligands or identifies multiple ligand fragments in the binding site. Our binding affinity predictor is capable of predicting the affinity of a given protein-ligand pair, requiring only limited information about the ligand pose. This enables screening without the costly step of first docking candidate molecules. Our framework will facilitate the design of ligands based on the targets surface information. It may significantly reduce the experimental screening load and ultimately reveal novel chemical compounds for targeting challenging proteins.


title: Self-similar tip growth links exocytosis profile with cell wall shape

abstract: Exocytosis plays a crucial role in regulating the growth and migration of filamentous tip-growing cells. We present a mathematical framework that infers the spatial profile of exocytosis from the cell morphology in self-similar growing cells that elongate while preserving their apical domain shapes. By applying the framework to cell wall outline data from experiments across walled cell species, we find that while tapered cells have their exocytosis concentrated at the apex, cells with flatter tip shape beyond a threshold require exocytosis to peak in an annulus region away from the apex.


title: Micro-CT analyses of the lung in mice: Parameters influencing the radiation dose and acquisition quality

abstract: Microcomputed tomography (micro-CT) is a frequently used imaging tool for a wide spectrum of in-vivo mouse models in basic and translational research. To allow an accurate interpretation of micro-CT images, high spatial resolution is necessary. However, this may also lead to a high radiation exposure of the animals. Therefore, animal welfare requires exact information about the expected radiation doses for experimental planning. To gain this, a mouse cadaver was herein used for micro-CT analyses under different conditions. For each radiation dose measurement, the cadaver was labeled with thermoluminescent dosimeter chips around the thoracic skin surface. Micro-CT scans of the thorax were performed with spatial resolutions of 35 {micro}m, 18 {micro}m and 9 {micro}m in combination with Al0.5, Al1.0, CuAl and Cu filters. As a surrogate of image quality, the number of identifiable lung vessels was counted on a transversal micro-CT slice. Measured radiation doses varied from 0.09 Gy up to 5.18 Gy dependent on resolution and filter settings. A significant dose reduction of > 75% was achieved by a Cu filter when compared to an Al0.5 filter. However, this resulted in a markedly reduced image quality and interpretability of microstructures due to higher radiation shielding and lower spatial resolution. Thus, the right combination of distinct filters and several scan protocol settings adjusted to the individual requirements can significantly reduce the radiation dose of micro-CT leading to a higher animal welfare standard.


title: Gestational Early-Time Restricted Feeding Results in Sex-Specific Glucose Intolerance in Adult Male Mice

abstract: The timing of food intake is a novel dietary component that can impact health. Time-restricted feeding (TRF), a form of intermittent fasting, manipulates food timing. During pregnancy, one may experience disruptions to food intake for diverse reasons (e.g. nausea and vomiting of pregnancy, food insecurity, desire to manage gestational weight gain, disordered eating behaviors, changes in taste and food preferences, etc) and therefore may experience periods of intentional or unintentional fasting similar to TRF protocols. Because interest in TRF is gaining popularity and feeding may be interrupted in those who are pregnant, it is important to understand the long-term effects of TRF during pregnancy on the resultant offspring. Using a mouse model, we tested the effects of gestational exposure to early TRF (eTRF) over the life course of both male and female offspring. Offspring body composition was similar between experimental groups in both males and females from weaning (day 21) to adulthood (day 70), with minor increases in food intake in eTRF females and improved glucose tolerance in males. After 10 weeks of high fat, high sucrose diet feeding, male eTRF offspring were more sensitive to insulin but developed glucose intolerance with impaired insulin secretion. As such, gestational eTRF causes sex-specific deleterious effects on glucose homeostasis after chronic high fat, high sucrose diet feeding in male offspring. Further studies are needed to determine the effect gestational eTRF has on the male pancreas as well as to elucidate the mechanisms that protect females from this metabolic dysfunction.


title: Interplay between R2R3 MYB-type activators and repressors regulates proanthocyanidin biosynthesis in banana (Musa acuminata)

abstract:
O_LIProanthocyanidins are oligomeric flavonoid pigments that promote plant disease resistance and benefit human health. However, the transcriptional regulatory network that fine-tunes proanthocyanidin biosynthesis in banana (Musa acuminata) fruit remains poorly understood.
C_LIO_LIWe characterized two proanthocyanidin-specific R2R3 MYB activators (MaMYBPA1-MaMYBPA2) and four repressors (MaMYBPR1-MaMYBPR4) to elucidate the mechanisms underlying the transcriptional regulation of proanthocyanidin biosynthesis in banana.
C_LIO_LIHeterologous expression of MaMYBPA1 and MaMYBPA2 partially complemented the Arabidopsis thaliana proanthocyanidin-deficient transparent testa2 mutant. MaMYBPA1 and MaMYBPA2 interacted physically with MaMYCs and bound the promoters of the genes encoding anthocyanin synthase, leucoanthocyanidin reductase, and anthocyanidin reductase in vitro and form functional MBW complexes with MaTTG1 in A. thaliana protoplasts to transactivated these promoters in vivo.
C_LIO_LIOverexpression of MaMYBPAs alone or with MaMYC in banana fruits induced proanthocyanidin accumulation and the transcription of proanthocyanidin biosynthesis- related genes. MaMYBPR repressors are also shown to interact with MaMYCs, forming repressing MBW complexes, and diminished proanthocyanidin accumulation. Interestingly the overexpression of MaMYBPA induces the expression of MaMYBPR, indicating an agile regulation of proanthocyanidin biosynthesis via the formation of competitive MBW complex. Taken together, our results reveal regulatory modules of R2R3 MYB- that fine-tune proanthocyanidin biosynthesis and offer possible targets for genetic manipulation in banana.
C_LI

category: plant biology


title: StandardRat: A multi-center consensus protocol to enhance functional connectivity specificity in the rat brain

abstract: Task-free functional connectivity in animal models provides an experimental framework to examine connectivity phenomena under controlled conditions and allows comparison with invasive or terminal procedures. To date, animal acquisitions are performed with varying protocols and analyses that hamper result comparison and integration. We introduce StandardRat, a consensus rat functional MRI acquisition protocol tested across 20 centers. To develop this protocol with optimized acquisition and processing parameters, we initially aggregated 65 functional imaging datasets acquired in rats from 46 centers. We developed a reproducible pipeline for the analysis of rat data acquired with diverse protocols and determined experimental and processing parameters associated with a more robust functional connectivity detection. We show that the standardized protocol enhances biologically plausible functional connectivity patterns, relative to pre-existing acquisitions. The protocol and processing pipeline described here are openly shared with the neuroimaging community to promote interoperability and cooperation towards tackling the most important challenges in neuroscience.


title: Retinotopic organization of feedback projections in primate early visual cortex: implications for active vision

abstract: Feedback connections play a major role in many theories of brain function. Previous studies of feedback connections to early visual areas have mainly concentrated on the representation of central visual fields. Here, injections of tracers at different eccentricities in areas V1 and V2 revealed retinotopically organized feedback. Peripheral injections revealed projections from 15 areas that are not labeled by central injections. Connection strengths for the majority of projections vary significantly with eccentricity in a systematic fashion with respect to distance and origin; whereas projections to central and upper visual fields are significantly stronger from ventral stream areas, peripheral and lower field projections are stronger from the dorsal stream. Non-invasive functional connectivity suggests a similar anatomical organization in humans. These features are discussed with respect to the cognitive and perceptual roles of these feedback pathways.


title: Reversible and gain modulation of neuronal responses and sensorimotor behavior by mid-infrared stimulation

abstract: Neuromodulation serves as a cornerstone for brain sciences and clinical applications. Mid-infrared stimulation (MIRS) has been recently reported to cause non-thermal modulation of brain functions. However little knowledge of mechanisms hampers its application. Here we bridge across ion channels, neuronal signals, and behavioral performances associated with sensorimotor transformation to provide evidence of how the alternation of neuronal activity by MIRS guides the change of behavioral performance in awake-behaving pigeons. We compared effects on visually-guided eye movements by applying MIRS and electrical stimulation (ES) in the pretectal nucleus lentiformis mesencephali (nLM). Distinct from ES, we found a specific gain modulation of MIRS to alter behavior in a manner of the strength of visual inputs. Our simultaneous extracellular recordings showed that MIRS can excite and inhibit the neuronal activity in the same pretectal neuron based on its ongoing sensory responsiveness levels in awake-behaving animals. We further applied computational simulations and found that MIRS can modulate the carbonyl group (-C=O) enriched on the potassium channel to resonate, and could affect action potential generation, alter neuronal responses to sensory inputs and then guide behavior. Our findings suggest that MIRS could be a promising approach for brain researches and neurological diseases, with gene free manipulation.


title: Blood-brain barrier dysfunction promotes astrocyte senescence through albumin-induced TGFβ signaling activation

abstract: Blood-brain barrier dysfunction (BBBD) and accumulation of senescent astrocytes occur during brain aging and contribute to neuroinflammation and disease. Here, we explored the relationship between these two age-related events, hypothesizing that chronic hippocampal exposure to the blood-borne protein serum albumin could induce stress-induced premature senescence (SIPS) in astrocytes via transforming growth factor beta 1 (TGF{beta}) signaling. We found that one week of albumin exposure significantly increased TGF{beta}1 and senescence marker expression in cultured rat hippocampal astrocytes. These changes were preventable by pharmacological inhibition of the type I TGF{beta} receptor (TGF{beta}R) ALK5. To study these effects in vivo, we utilized an animal model of BBBD in which albumin was continuously infused into the lateral ventricles of adult mice. Consistent with our in vitro results, one week of albumin infusion significantly increased TGF{beta} signaling activation and the burden of senescent astrocytes in hippocampal tissue. Pharmacological inhibition of TGF{beta}R ALK5 or conditional genetic knockdown of astrocytic TGF{beta}R prior to albumin infusion was sufficient to prevent albumin-induced astrocyte senescence. Together, these results establish a link between TGF{beta} signaling activation and astrocyte senescence and suggest that prolonged exposure to serum albumin due to BBBD can trigger these phenotypic changes.


title: Downregulating PTBP1 fails to convert astrocytes into hippocampal neurons and to alleviate symptoms in Alzheimer's mouse models

abstract: Conversion of astroglia into functional neurons has been considered as a promising therapeutic strategy for neurodegenerative diseases. Recent studies reported that downregulation of the RNA binding protein, PTBP1, converts astrocytes into neurons in situ in multiple mouse brain regions, consequently improving pathological phenotypes associated with Parkinsons disease, RGC loss, and aging. Here, we demonstrate that PTBP1 downregulation using antisense oligonucleotides or an astrocyte specific AAV-mediated shRNA system fails to convert hippocampal astrocytes into neurons in WT, and {beta}-amyloid (5xFAD) and tau (PS19) Alzheimers disease (AD) mouse models, and fails to reverse synaptic/cognitive deficits and AD-associated pathology. Similarly, PTBP1 downregulation cannot convert astrocytes into neurons in the striatum and substantia nigra. Together, our study suggests that cell fate conversion strategy for neurodegenerative disease therapy through manipulating one single gene, such as PTBP1, warrants more rigorous scrutiny.


title: Testosterone eliminates strategic prosocial behavior

abstract: Humans are strategically more prosocial when their actions are being watched than when they act alone. Using a psychopharmacogenetic approach, we investigated the computational and endocrinological mechanisms of such audience-driven prosociality. 187 participants received either a single dose of testosterone or a placebo and performed a prosocial and self-oriented reinforcement learning task. Crucially, the task was performed either in private or when being watched. Rival theories of testosterones role in status-seeking suggest that the hormone might either strengthen or diminish audience-depended generosity. We show that exogenous testosterone strongly decreases submission to audience expectations, full eliminating strategic i.e., feigned generosity. We next performed reinforcement-learning drift-diffusion modeling to elucidate which latent aspects of decision-making testosterone acted on. Computational modeling revealed that testosterone compared to placebo did not deteriorate reinforcement learning per se, rather, in presence of the audience, the hormone impacted the expression of the learned information into behavioral choice. These results indicate that instead of deceptively increasing socially desirable behavior, testosterone boosts honest forms of status-seeking, arguably by impacting the motivational link between learned values and behavior.


title: The Toll and Imd pathway, the complement system and lectins during immune response of the nemertean Lineus ruber

abstract: Innate immunity is the first line of defense against pathogens. In animals, the Toll pathway, the Imd pathway, the complement system, and lectins are well-known mechanisms involved in innate immunity. Although these pathways and systems are well understood in vertebrates and arthropods, they are understudied in other invertebrates. In order to shed light on immunity in the nemertean Lineus ruber, we performed a transcriptomic survey and identified the main components of the Toll pathway (e.g. myD88, dorsal/dif/NF{kappa}B-p65), the Imd pathway (e.g. imd, relish/NF{kappa}B-p105/100), the complement system (e.g. C3, cfb) and some lectins (FreD-Cs and C-lectins). In situ hybridization showed that TLR{beta}1, TLR{beta}2 and imd and are expressed in the nervous system, the complement gene C3-1 is expressed in the gut and the lectins in the nervous system, the blood, and the gut. To reveal their potential role in defense mechanisms, we performed immune challenge experiments, in which Lineus ruber specimens were exposed to the gram-negative bacteria Vibrio diazotrophicus. Our results show the upregulation of specific components of the Toll pathway (TLR3, TLR{beta}1, and TLR{beta}2), the complement system (C3-1), and lectins (c-lectin2 and fred-c5). Therefore, similarly to what occurs in other invertebrates, our study shows that components of the Toll pathway, the complement system and lectins are involved in the immune response in the nemertean Lineus ruber. The presence of these pathways and systems in Lineus ruber, but also in other spiralians, in protostomes and in deuterostomes suggest that these pathways and systems were involved in the immune response in the stem species of Bilateria.

category: immunology


title: Correlative light and electron microscopy reveals fork-shaped structures at actin entry sites of focal adhesions

abstract: Focal adhesions (FAs) are the main cellular structures to link the intracellular cytoskeleton to the extracellular matrix. FAs mediate cell adhesion, are important for cell migration and are involved in many (patho)-physiological processes. Here we examined FAs and their associated actin fibres using correlative fluorescence and scanning electron microscopy (SEM). We used fluorescence images of cells expressing paxillin-GFP to define the boundaries of FA complexes in SEM images, without using SEM contrast enhancing stains. We observed that SEM contrast was increased around the actin fibre entry site in 98% of FAs, indicating increases in protein density and possibly also phosphorylation levels in this area. In nearly three quarters of the FAs, these nanostructures had a fork shape, with the actin forming the stem and the high contrast FA areas the fork. In conclusion, the combination of fluorescent and electron microscopy allowed accurate localisation of a highly abundant, novel fork structure at the FA-actin interface.

category: cell biology


title: Improved imaging and preservation of lysosome dynamics using silver nanoparticle-enhanced fluorescence

abstract: The dynamics of living cells can be studied by live-cell fluorescence microscopy. However, this often requires the use of excessive light energy to obtain good signal-to-noise ratio, which can then photobleach the fluorochromes used, and more worrisome, lead to photo-toxicity. Thus, strategies that can reduce the amount and/or exposure to excitation light would improve live-cell microscopy. Upon light excitation, noble metal nanoparticles such as silver (AgNP) generate plasmons, which can amplify the excitation of fluorophores that are proximal to the surface of the nanoparticle. These can also couple to the oscillating dipole of nearby radiating fluorophores, which modifies the rate of emission and enhances their fluorescence. Here, we show that AgNP fed to cells to accumulate within lysosomes enhanced the fluorescence of lysosome-targeted BODIPY-cholesterol and DQ-BSA. Moreover, AgNP increased the fluorescence of GFP fused to the cytosolic tail of LAMP1, showing that metal enhanced fluorescence occurs across the lysosomal membrane. Importantly, by using AgNP, we could track lysosome motility with reduced laser power without damaging and altering lysosome dynamics. Overall, AgNP-enhanced fluorescence may be a useful tool to study the dynamics of the endo-lysosomal pathway while minimizing photo-toxicity.


title: Dynamic Landscapes of tRNA Transcriptomes and Translatomes in Diverse Mouse Tissues

abstract: Although the function of tRNA in translational process is well established, it remains controversial whether tRNA abundance is tightly associated with translational efficiency (TE) in mammals. For example, how critically the expression of tRNAs contributes to the establishment of tissue-specific proteomes in mammals has not been well addressed. Here, we measured both tRNA expression using DM-tRNA-seq and ribosome-associated mRNAs in the brain, heart, and testis of RiboTag mice. Remarkable variation in the expression of tRNA isodecoders was observed among the different tissues. When the statistical effect of isodecoder-grouping on reducing variations is considered through permutating the anticodons, we observed an expected reduction in the tissue-variations of anticodon expression, an unexpected smaller variation of anticodon usage bias, and an unexpected larger variation of tRNA isotype expression. Regardless whether or not they share the same anticodons, isotypes encoding the same amino acids are co-expressed across different tissues. Based on the tRNA expression and TE computed from RiboTag-seq, we find that the tRNA adaptation index (tAI) values and TE are significantly correlated in the same tissues but not among tissues; tRNAs and the amino acid compositions of translating peptides are positively correlated in the same tissues but not between tissues. We therefore hypothesize that the tissue-specific expression of tRNAs might be related to post-transcriptional mechanisms, such as aminoacylation, modification, and tRNA-derived small RNAs (tsRNAs). This study provides a resource for tRNA and translation studies to gain novel insights into the dynamics of tRNAs and their role in translational regulation.


title: The Image Data Explorer: interactive exploration of image-derived data

abstract: Many bioimage analysis projects produce quantitative descriptors of regions of interest in images. Associating these descriptors with visual characteristics of the objects they describe is a key step in understanding the data at hand. However, as many bioimage data and their analysis workflows are moving to the cloud, addressing interactive data exploration in remote environments has become a pressing issue. To address it, we developed the Image Data Explorer (IDE) as a web application that integrates interactive linked visualization of images and derived data points with exploratory data analysis methods, annotation, classification and feature selection functionalities. The IDE is written in R using the shiny framework. It can be easily deployed on a remote server or on a local computer. The IDE is available at https://git.embl.de/heriche/image-data-explorer and a cloud deployment is accessible at https://shiny-portal.embl.de/shinyapps/app/01_image-data-explorer.


title: Coordination between ECM and cell-cell adhesion regulates the development of islet aggregation, architecture, and functional maturation

abstract:
Pancreatic islets are 3-dimensional cell aggregates consisting of unique cellular composition, cell-to-cell contacts, and interactions with blood vessels. Cell aggregation is essential for islet endocrine function; however, it remains unclear how developing islets establish aggregation. By combining genetic animal models, imaging tools, and gene expression profiling, we demonstrate that the islet aggregation is regulated by extracellular matrix signaling and cell-cell adhesion. Islet endocrine cell-specific inactivation of extracellular matrix receptor Integrin {beta}1 disrupted blood vessel interactions but promoted cell-cell adhesion and the formation of larger islets. In contrast, ablation of cell-cell adhesion molecule -Catenin promoted blood vessel interactions yet compromised islet clustering. Simultaneous removal of Integrin {beta}1 and -Catenin disrupts islet aggregation and the endocrine cell maturation process, demonstrating that establishment of islet aggregates is essential for functional maturation. Our study provides new insights into understanding the fundamental self-organizing mechanism for islet aggregation, architecture, and functional maturation.

HIGHLIGHTSO_LIIslet vascularization and aggregation are regulated via ECM-Itgb1 signaling
C_LIO_LIECM-Itgb1 signaling negatively controls islet aggregation via regulation of cell-cell adhesion during development
C_LIO_LICell-cell adhesion negatively regulates the interaction of endocrine cell-vasculature in islets
C_LIO_LIDifferential cell adhesion regulates the establishment of islet architecture
C_LIO_LIEndocrine functional maturation depends on islet aggregation regulated by the coordination of ECM-Itgb1 signaling and cell-cell adhesion
C_LI


title: Isthmal stem cells sustain intestinal homeostasis and regeneration

abstract: The currently accepted intestinal epithelial cell organization model equates crypt base columnar (CBC) cells, marked by high levels of Lgr5 expression, with the intestinal stem cell (ISC)1. However, recent intestinal regeneration studies have uncovered limitations of the  Lgr5-CBC model2, 3, leading to two major views: one favoring the presence of a quiescent reserve stem cell population4-7, the other calling for differentiated cell plasticity8-11. To test if an alternative model may help reconcile these perspectives, we studied the hierarchical organization of crypt epithelial cells in an unbiased fashion, by combining high-resolution, single-cell profiling and lineage tracing in multiple transgenic mouse models. These show that Lgr5 is not a specific ISC marker; rather, cells located in the crypt isthmus, which include Lgr5low cells, comprise the ISCs that sustain tissue homeostasis. Following irradiation or intestinal injury, surviving ISCs and progenitors, but not differentiated cells, participate in intestinal regeneration, suggesting that neither de-differentiation nor reserve stem cell populations are drivers of intestinal regeneration. Our results provide a novel viewpoint for the intestinal crypt epithelium, in which ISCs localize to the crypt isthmus, and ISC potential is restricted to stem and progenitor cells.


title: Digital whole-community phenotyping to assess morphological and physiological features of plant communities in the field

abstract: Traits link observable patterns in plants to ecosystem functions and processes and help to derive general rules and predictions about responses to environmental gradients, global change and perturbations. Ecological field studies often use manual low-throughput methods to assess plant phenotypes and integrate species-specific traits to community-wide indices. In contrast, greenhouse or lab-based studies, mostly in agriculture, employ high-throughput phenotyping for plant individuals to track their growth or fertilizer and water demand. We customized an automated plant phenotyping system (PlantEye F500, Phenospex, Heerlen, The Netherlands) for its mobile application in the field for digital whole-community phenotyping (DWCP). By scanning whole plant communities, we gather, within seconds and non-invasively, multispectral and physiological information while simultaneously capturing the 3-dimensional structure of the vegetation. We demonstrated the potential of DWCP by tracking plant community responses to experimental land-use treatments over two years. DWCP captured short- and long-term changes in morphological and physiological plant community properties in response to mowing and fertilizer treatments and thus reliably informed about changes in land-use. In contrast, manually measured community-weighted mean traits and species composition remained largely unaffected and were not informative about these treatments. Thus, DWCP proved to be an efficient method to measure morphological and physiological characteristics of plant communities, complements other methods in trait-based ecology, provides indicators of ecosystem states, and may help to forecast tipping points in plant communities often associated with irreversible changes in ecosystems.


title: Which life history strategy can maintain high genetic diversity in plants?

abstract: Classifying a variety of plant life histories is an important step to understand ecological and evolutionary background of life history diversification in plants. Principal component analysis (PCA) of life history traits, as well as elasticity analysis using a ternary diagram, successfully categorized the diverse life histories and clarified their link to various ecological characteristics, such as population dynamics, functional traits, and conservation status. However, the relationships with population genetic properties, including genetic diversity, have not been explored very much. In this study, we overlaid annual change rate of expected heterozygosity{eta} on life history spectrum obtained by PCA and elasticity analysis to examine which life history strategy can maintain high genetic diversity over time. We found that{eta} gradually changed along the fast-slow continuum and that slow-paced life histories maintained high genetic diversity, probably due to generation overlap. Elasticity analysis showed that life histories that highly depend on stasis also maintained genetic diversity. As genetic diversity reflects adaptive potential to environmental changes, our study provides a new genetic perspective on adaptative evolution and population viability to life history study.


title: Toward Quantitative Metabarcoding

abstract: Amplicon-sequence data from environmental DNA (eDNA) and microbiome studies provides important information for ecology, conservation, management, and health. At present, amplicon-sequencing studies - known also as metabarcoding studies, in which the primary data consist of targeted, amplified fragments of DNA sequenced from many taxa in a mixture - struggle to link genetic observations to underlying biology in a quantitative way, but many applications require quantitative information about the taxa or systems under scrutiny. As metabarcoding studies proliferate in ecology following decades of microbial and microbiome work using similar techniques, it becomes more important to develop ways ot make them quantitative to ensure that their conclusions are adequately supported. Here we link previously disparate sets of techniques for making such data quantitative, showing that the underlying PCR mechanism explains observed patterns of amplicon data in a general way. By modeling the process through which amplicon-sequence data arises, rather than transforming the data post-hoc, we show how to estimate the starting DNA proportions from a mixture of many taxa. We illustrate how to calibrate the model using mock communities and apply the approach to simulated data and a series of empirical examples. Our approach opens the door to improve the use of metabarcoding data in a wide range of applications in ecology, public health, and related fields.


title: Novel trans-translation-associated gene regulation revealed by prophage excision-triggered switching of ribosome rescue pathway

abstract: Escherichia coli has multiple pathways to release a nonproductive ribosome complex stalled at the 3 end of nonstop mRNAs: SsrA RNA-mediated trans-translation and stop codon-independent termination by ArfA/RF2 or ArfB (YaeJ). The arfA mRNA lacks a stop codon and thus its expression is repressed by trans-translation. Therefore, ArfA is considered to complement the ribosome rescue activity of trans-translation, but the situations in which ArfA is expressed to rescue the nonproductive complexes have not been elucidated. Here, we demonstrated that the excision of the CP4-57 prophage adjacent to the E. coli ssrA gene leads to the inactivation of SsrA RNA and switches the primary rescue pathway from trans-translation to the ArfA/RF2 pathway. A comparative quantitative proteomic analysis revealed that the switching of the rescue pathway rearranges not only the proteome landscape in E. coli cells but also the phenotype, such as motility. Among the proteins with significantly increased abundance in the ssrA-inactivated cells, we found ZntR, whose mRNA is transcribed together as the upstream part of the nonstop arfA mRNA. Further analysis revealed that the translation of the nonstop ORF of arfA triggered the repression of the upstream zntR ORF, via trans-translation-coupled exonucleolytic (RUTEX) degradation by a polynucleotide phosphorylase. These results provide a novel example of trans-translation-dependent regulation, and shed new light on the physiological roles of prophages in gene expression.

category: genetics


title: happi: a Hierarchical Approach to Pangenomics Inference

abstract: Recovering metagenome-assembled genomes (MAGs) from shotgun sequencing data is an increasingly common task in microbiome studies, as MAGs provide deeper insight into the functional potential of both culturable and non-culturable microorganisms. However, metagenome-assembled genomes vary in quality, and may contain omissions and contamination. These errors present challenges for detecting genes and comparing gene enrichment across sample types. To address this, we propose happi, an approach to testing hypotheses about gene enrichment that accounts for genome quality. We illustrate the advantages of happi over existing approaches using published Saccharibacteria MAGs and via simulation.


title: Museomic approaches to genotype historic Cinchona barks

abstract: Over the last few centuries, millions of plant specimens have been collected and stored within herbaria and biocultural collections. They therefore represent a considerable resource for a broad range of scientific uses. However, collections degrade over time, and it is therefore increasingly difficult to characterise their genetic signatures. Here, we genotyped highly degraded Cinchona barks and leaves from herbaria using two separate high-throughput sequencing methods (HtS) and compared their performance. We subsequently genotyped specimens using genome skimming, the most commonly performed high-throughput sequencing (HtS) technique. We additionally used a recently developed capture bait set (Angiosperm353) for a target enrichment approach. Specifically, phylogenomic analyses of modern leaf and historical barks of Cinchona were performed, including 23 historical barks and six fresh leaf specimens. We found that samples degraded over time, which directly reduced the quantity and quality of the data produced by both methodologies (in terms of reads mapped to the references). However, we found that both approaches generated enough data to infer phylogenetic relationships, even between highly degraded specimens that are over 230 years old. However, the target capture kit produced data for target nuclear loci and also chloroplast data, which allowed for phylogenies to be inferred from both genomes, whereas it was only possible to use chloroplast data using genome skimming. We therefore find the Angiosperms353 target capture kit a powerful alternative to genome skimming, which can be used to obtain more information from herbarium specimens, and ultimately additional cultural benefits.


title: Cost-efficient boundary-free surface patterning achieves high effective-throughput of time-lapse microscopy experiments

abstract: Time-lapse microscopy plays critical roles in the studies of cellular dynamics. However, to set up a time-lapse movie experiments is not only laborious but also with low output, mainly due to the cell-losing problem (i.e., cell moving out of limited field of view), especially in a long time recording. To overcome these issues, we have designed a cost-efficient way that enables cell patterning on the imaging surfaces without any physical boundaries. Using mouse embryonic stem cells as an example system, we have demonstrated that our boundary-free patterned surface solves the cell-losing problem without disturbing their cellular phenotype. Statistically, the presented system increases the effective-throughput of time-lapse microscopy experiments by order of magnitude.


title: Algorithmic design of 3D wireframe RNA polyhedra

abstract: We address the problem of de novo design and synthesis of nucleic acid nanostructures, a challenge that has been considered in the area of DNA nanotechnology since the 1980s and more recently in the area of RNA nanotechnology. Towards this goal, we introduce a general algorithmic design process and software pipeline for rendering 3D wireframe polyhedral nanostructures in single-stranded RNA. To initiate the pipeline, the user creates a model of the desired polyhedron using standard 3D graphic design software. As its output, the pipeline produces an RNA nucleotide sequence whose corresponding RNA primary structure can be transcribed from a DNA template and folded in the laboratory. As case examples, we design and characterize experimentally three 3D RNA nanostructures: a tetrahedron, a bipyramid and a prism. The design software is openly available, and also provides an export of the targeted 3D structure into the oxRNA molecular dynamics simulator for easy simulation and visualization.


title: Projecting clumped transcriptomes onto single cell atlases to achieve single cell resolution

abstract: Multi-modal single cell RNA assays capture RNA content as well as other data modalities, such as spatial cell position or the electrophysiological properties of cells. Compared to dedicated scRNA-seq assays however, they may unintentionally capture RNA from multiple adjacent cells, exhibit lower RNA sequencing depth compared to scRNA-seq, or lack genome-wide RNA measurements. We present scProjection, a method for mapping individual multi-modal RNA measurements to deeply sequenced scRNA-seq atlases to extract cell type-specific, single cell gene expression profiles. We demonstrate several use cases of scProjection, including the identification of spatial motifs from spatial transcriptome assays, distinguishing RNA contributions from neighboring cells in both spatial and multi-modal single cell assays, and imputing expression measurements of un-measured genes from gene markers. scProjection therefore combines the advantages of both multi-modal and scRNA-seq assays to yield precise multi-modal measurements of single cells.


title: ConsAlign: simultaneous RNA structural aligner based on rich transfer learning and thermodynamic ensemble model of alignment scoring

abstract:
MotivationTo capture structural homology in RNAs, predicting RNA structural alignments has been a fundamental framework around RNA science. Learning simultaneous RNA structural alignments in their rich scoring parameterization is an undeveloped subject because evaluating them is computationally expensive in nature.

ResultsWe developed ConsTrain--a gradient-based machine learning method for rich structural alignment scoring. We also implemented ConsAlign--a simultaneous RNA structural aligner composed of ConsTrains learned scoring parameters. To aim for better structural alignment quality, ConsAlign employs (1) transfer learning from well-defined scoring models and (2) the ensemble model between the ConsTrain model and a mature thermodynamic scoring model. Keeping comparable running time, ConsAlign demonstrated the best alignment prediction quality among current RNA structural aligners.

Availability and implementationOur code and our data are freely available at https://github.com/heartsh/consalign.

Contactheartsh@heartsh.io

Supplementary informationSupplementary data are available at Bioinformatics


title: Systematic Replication Enables Normalization of High-throughput Imaging Assays

abstract:
MotivationHigh-throughput fluorescent microscopy is a popular class of techniques for studying tissues and cells through automated imaging and feature extraction of hundreds to thousands of samples. Like other high-throughput assays, these approaches can suffer from unwanted noise and technical artifacts that obscure the biological signal. In this work we consider how an experimental design incorporating multiple levels of replication enables removal of technical artifacts from such image-based platforms.

ResultsWe develop a general approach to remove technical artifacts from high-throughput image data that leverages an experimental design with multiple levels of replication. To illustrate the methods we consider microenvironment microarrays (MEMAs), a high-throughput platform designed to study cellular responses to microenvironmental perturbations. In application on MEMAs, our approach removes unwanted spatial artifacts and thereby enhances the biological signal. This approach has broad applicability to diverse biological assays.

AvailabilityRaw data is on synapse (syn2862345), analysis code is on github (gjhunt/mema norm), a Docker image is available on dockerhub (gjhunt/memanorm). online.


title: Improved sequence mapping using a complete reference genome and lift-over

abstract: Complete, telomere-to-telomere genome assemblies promise improved analyses and the discovery of new variants, but many essential genomic resources remain associated with older reference genomes. Thus, there is a need to translate genomic features and read alignments between references. Here we describe a new method called levioSAM2 that accounts for reference changes and performs fast and accurate lift-over between assemblies using a whole-genome map. In addition to enabling the use of multiple references, we demonstrate that aligning reads to a high-quality reference (e.g. T2T-CHM13) and lifting to an older reference (e.g. GRCh38) actually improves the accuracy of the resulting variant calls on the old reference. By leveraging the quality improvements of T2T-CHM13, levioSAM2 reduces small-variant calling errors by 11.4-39.5% compared to GRC-based mapping using real Illumina datasets. LevioSAM2 also improves long-read-based structural variant calling and reduces errors from 3.8-11.8% for a PacBio HiFi dataset. Performance is especially improved for a set of complex medically-relevant genes, where the GRC references are lower quality. The software is available at https://github.com/milkschen/leviosam2 under the MIT license.