Large-scale identification of viral quorum sensing systems reveals density-dependent sporulation-hijacking mechanisms in bacteriophages Communication between viruses supported by quorum sensing systems (QSSs) were found to optimize the fitness of temperate bacteriophages of Bacilli by guiding the transition from the host-destructive lytic cycle to the host-protective lysogenic cycle in a density-dependent manner. All known phage-encoded QSSs consist of a communication propeptide and a cognate intracellular receptor that regulates the expression of adjacent target genes upon recognition of the matured peptide, a signature known as RRNPP and found in chromosomes, plasmids and phages of Firmicutes bacteria. Recently, we have introduced the RRNPP_detector software to detect novel genetic systems matching the RRNPP signature, which unearthed many novel phage-encoded candidate QSSs. Here, by looking at the adjacent genes likely regulated by these viral candidate QSSs, we identified an unsuspected clustering of viral QSSs with viral genes whose bacterial homologs are key regulators of the last-resort bacterial sporulation initiation pathway (rap, spo0E or abrB). Consistently, we found evidence in published data that certain of these QSSs encoded by prophages (phage genomes inserted within a bacterial genome) dynamically manipulate the timing of sporulation in the host. Because these viral QSSs are genetically diverse and are found associated with different sporulation regulators, this suggests a convergent evolution in bacteriophages of density-dependent sporulation-hijacking mechanisms.

SIGNIFICANCECommunication between viruses supported by quorum sensing systems (QSSs) is a brand new research area that has transformed our views of viral adaptation and virus-host co-evolution. The viral QSSs discovered so far were found to guide the lysis-lysogeny decision in temperate bacteriophages as a function of phage density. Here, we identified that quorum sensing-mediated communication between phages can not only guide the regulation of viral processes but also the manipulation of the bacterial sporulation pathway. Our finding introduces the new view that not only bacteria decide when it is time to sporulate, some bacteriophages are also key stakeholders in this dynamical decision-making process. Considering that spores are the transmissive form of many pathogens, these new insights have important applied implications.
Minimizing spectral overlap in multicolor flow cytometry experiments Selecting fluorochromes for polychromatic panels for flow cytometry is complex and time-consuming. Poorly designed panels can result in overlap between the emission spectra of the different fluorochromes, making their signals difficult to separate. While assessing all possible panels is simple to do programmatically, the combinatorial complexity of most real world problems renders brute force computation impractical. Here we present a novel complexity optimization algorithm for fast design of minimal spectral overlap fluorochrome combinations. To aid researchers in designing fluorochrome panels we implemented the algorithm in a web server, Spectracular, that also considers instrument laser configuration and allows users to define various constraints related to antibody-fluorochrome availability and marker co-expression.
Relationship between vestibular hair cell loss and deficits in two anti-gravity reflexes in the rat The tail-lift reflex and the air-righting reflex in rats are anti-gravity reflexes that depend on vestibular function. To begin identifying their cellular basis, this study examined the relationship between reflex loss and the graded lesions caused in the vestibular sensory epithelia by varying doses of an ototoxic compound. After ototoxic exposure, we recorded these reflexes using high speed video. The movies were used to obtain objective measures of the reflexes: the minimum angle formed by the nose, the back of the neck and the base of the tail during the tail-lift maneuver and the time to right in the air-righting test. The vestibular sensory epithelia were then collected from the rats and used to estimate the loss of type I (HCI), type II (HCII) and all hair cells (HC) in both central and peripheral parts of the crista, utricle, and saccule. As expected, tail-lift angles decreased, and air-righting times increased, while the numbers of HCs remaining in the epithelia decreased in a dose-dependent manner. The results demonstrated greater sensitivity of HCI compared to HCII to the IDPN ototoxicity, as well as a relative resiliency of the saccule compared to the crista and utricle. Comparing the functional measures with the cell counts, we observed that loss of the tail-lift reflex associates better with HCI than with HCII loss. In contrast, most HCI in the crista and utricle were lost before air-righting times increased. These data suggest that these reflexes depend on the function of non-identical populations of vestibular HCs. Liberation from fibrogenesis or tumorigenesis via cellular senescence by a novel small molecule BackgroundUncontrolled proliferative diseases such as fibrosis or cancer can be fatal. We previously found that a compound containing a chromone scaffold, ONG41008, had potent anti-fibrotic effects in diseased human lung myofibroblasts but not normal human lung fibroblasts.

MethodsWe investigated the effects of ONG41008 on tumor cells, and compared these effects with those in pathogenic myofibrotic cells and normal fibroblasts cells.

FindingsStimulation of A549 lung carcinoma epithelial cells with ONG41008 resulted in cellular senescence, indicating that dysregulated cell proliferation is common to fibrotic cells and tumor cells. Replicative senescence of A549 cells resulted in multinucleation, which was followed by oncogene-induced senescence. There was significant upregulation of expression and nuclear translocation of p-TP53 and p16 in ONG41008-treated A549 cells, and all cells died after 72 hr. Similar effects occurred after ONG41008 treatment in several human aggressive cancer cell lines such as PANC1, MCF7, PC3, or primary non-small cell lung carcinoma cells. Unlike cisplatin, ONG41008 was not toxic to normal human lung fibroblasts or primary prostate epithelial cells, suggesting ONG41008 can distinguish the intracellular microenvironment between normal cells and aged or diseased cells. This effect might occur as a result of the increased NAD/NADH ratio or increased lactate dehydrogenase levels in aged or diseased cells.

InterpretationTo our best knowledge, this is the first study to show that a small molecule can arrest uncontrolled proliferation during fibrogenesis or tumorigenesis in vitro. ONG41008 could be a potential drug for a broad range of fibrotic or tumorigenic diseases.

Research in ContextO_ST_ABSEvidence before this studyC_ST_ABSThe notion that aging is a disease and that diseases occur as a consequence of aging was first put forward by David Sinclair and colleagues (Aging Cell;14(4):497-510). There are biological systems that provide evidence for this notion; for example, somatic cells can revert to embryonic cells, producing younger somatic cells. This phenomenon underlies induced pluripotent stem cells. Another example is that some types of jellyfish can live forever. These examples suggest that a counter-aging program exists in animals. Human diseases are the manifestations of cell aging generated by the accumulation of somatic mutations. Aged and pathogenic cells are senescent, so a drug that specifically targeted senescent cells might initiate a cellular program that could ameliorate age-associated disease. Indeed, the kinase inhibitor dasatinib induces cellular senescence (Clin Ther 2007 29:2289-2308). In 2017, two drugs that target senescent cells were identified: quercetin and fisetin. These drugs selectively kill senescent cells, and are referred to as senolytic drugs or senotherapeutics (Aging 2017 8;9(3):955-963). Although it is established that senescent cells accumulate in cancer and idiopathic pulmonary fibrosis (Nat Commun 2017 23;8 :14532), the effect of senolytic drugs in these diseases is largely unknown.

Added value of this studyThis study characterized a novel drug, termed ONG41008, which was found to have both senogenic and senolytic effects in cell-based assays. ONG41008 induced senescence in myofibroblasts and several cancer cell lines representative of aggressive human cancers, which was followed by cell death. Importantly, ONG41008 exhibited essentially no toxicity on normal human lung fibroblasts or primary prostate epithelial cells.

Implication of all the evidenceBased on our results, we believe that ONG41008 is a potent inducer of cellular senescence (replicative senescence and oncogene-induced senescence) and causes arrest of uncontrolled, pathogenic proliferation of myofibroblasts or cancer cells. Chromosome breaks in breast cancers occur near herpes tumor virus sequences and explain why the cancer comes back One explanation for why breast cancer has a relentless tendency to come back after treatment is that breast cancer phenomes are fluid, so it is difficult for therapy to target new phenotypes that arise. In breast cancer, viral interactions cause chromosome breakage and fusion, producing anaphase bridges containing two centromeres. Cell division then leads to more chromosome breaks, more mutations, and new cancer phenotypes. Analyses of public data from about 2100 breast cancers implicate Epstein-Barr virus (EBV or Human Herpes Virus 4) in the cycles that break chromosomes and produce anaphase bridges. EBV generates breakage cycles with highly fragmented chromosomes (chromothripsis) as an inherent feature. These episodes continually produce new cancer driver mutations. DNA at chromothripsis boundaries in breast cancers consistently matches EBV tumor variants. Breakpoints are often near known EBV binding sites, and breast cancer shares an EBV methylation signature with other known EBV cancers. Breast cancer breakpoints on all chromosomes cluster around the same positions as in cancers definitively associated with EBV infection (nasopharyngeal cancer and Burkitts lymphoma). The status of EBV in the microbiome and the presence of anaphase bridges are phenotypic markers of breast cancer that are not explicitly targeted by chemotherapy. The breast cancer phenome continually supplies new cancer driver genes that foster breast cancer recurrence.

SummaryBreaks in breast cancer chromosomes and in fragments of anaphase bridges occur mainly near regions that resemble Epstein-Barr virus (EBV) tumor variants

Extensive breaks are an inherent outcome of anaphase bridges and the reassembly of highly fragmented chromosomes continually generates new cancer driver mutations and new cancer phenotypes.

Treatment does not explicitly target the underlying breakage and reassembly cycles so breast cancer often comes back.

The breast cancer phenome is not fixed and can continuously generate new phenotypes

Graphical Abstract

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org.highwire.dtl.DTLVardef@6537cforg.highwire.dtl.DTLVardef@132fa50org.highwire.dtl.DTLVardef@1cfcb04org.highwire.dtl.DTLVardef@11979aa_HPS_FORMAT_FIGEXP  M_FIG C_FIG Darwin's small and medium ground finches might have taste preferences, but not for human foods Urbanization is rapidly changing ecological niches. On the inhabited Galapagos Islands, Darwins finches consume human-introduced foods preferentially; however, it remains unclear why. Here we presented pastry with flavour profiles typical of human foods (oily, salty, sweet) to small ground finches (Geospiza fuliginosa) and medium ground finches (Geospiza fortis) to test if latent taste preferences might drive selection of human foods. If human-food flavours were consumed more than a neutral or bitter control only at sites with human foods, then we predicted tastes were acquired after urbanization; however, if no site-differences were found then this would indicate latent taste preferences. Contrary to both predictions, we found little evidence that human-food flavours were preferred compared to control flavours at any site. Instead, finches showed a weak aversion to oily foods, but only at remote (no human foods present) sites. This was further supported by behavioural responses, with beak wiping occurring more often at remote sites after finches tasted flavours associated with human foods. Our results suggest, therefore, that while Darwins finches may have acquired a tolerance to the flavours of human food, latent taste preferences are unlikely to have played a major role in their dietary response to increased urbanization. Improving human coronavirus OC43 (HCoV-OC43) research results comparability in studies using HCoV-OC43 as a surrogate for SARS-CoV-2 The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has renewed interest in human coronaviruses that cause the common cold, particularly as research with them at biosafety level (BSL)-2 avoids the added costs and biosafety concerns that accompany work with SARS-COV-2, BSL-3 research. One of these, human coronavirus OC43 (HCoV-OC43), is a well-matched surrogate for SARS-CoV-2 because it is also a Betacoronavirus, targets the human respiratory system, is transmitted via respiratory aerosols and droplets and is relatively resistant to disinfectants. Unfortunately, growth of HCoV-OC43 in the recommended human colon cancer (HRT-18) cells does not produce obvious cytopathic effect (CPE) and its titration in these cells requires expensive antibody-based detection. Consequently, multiple quantification approaches for HCoV-OC43 using alternative cell lines exist, which complicates comparison of research results. Hence, we investigated the basic growth parameters of HCoV-OC43 infection in three of these cell lines (HRT-18, human lung fibroblasts (MRC-5) and African green monkey kidney (Vero E6) cells) including the differential development of cytopathic effect (CPE) and explored reducing the cost, time and complexity of antibody-based detection assay. Multi-step growth curves were conducted in each cell type in triplicate at a multiplicity of infection of 0.1 with daily sampling for seven days. Samples were quantified by tissue culture infectious dose50(TCID50)/ml or plaque assay (cell line dependent) and additionally analyzed on the Sartorius Virus Counter 3100 (VC), which uses flow virometry to count the total number of intact virus particles in a sample. We improved the reproducibility of a previously described antibody-based detection based TCID50 assay by identifying commercial sources for antibodies, decreasing antibody concentrations and simplifying the detection process. The growth curves demonstrated that HCoV-O43 grown in MRC-5 cells reached a peak titer of [~]107 plaque forming units/ml at two days post infection (dpi). In contrast, HCoV-OC43 grown on HRT-18 cells required six days to reach a peak titer of [~]106.5 TCID50/ml. HCoV-OC43 produced CPE in Vero E6 cells but these growth curve samples failed to produce CPE in a plaque assay after four days. Analysis of the VC data in combination with plaque and TCID50 assays together revealed that the defective:infectious virion ratio of MRC-5 propagated HCoV-OC43 was less than 3:1 for 1-6 dpi while HCoV-OC43 propagated in HRT-18 cells varied from 41:1 at 1 dpi, to 329:4 at 4 dpi to 94:1 at 7 dpi. These results should enable better comparison of extant HCoV-OC43 study results and prompt further standardization efforts. Epitope-tagging of the endogenous murine BiP/GRP78/Hspa5 locus allows direct analysis of the BiP interactome and protein misfolding in vivo BiP/GRP78, encoded by the Hspa5 gene, is the major HSP70 family member in the endoplasmic reticulum (ER) lumen, and controls ER protein folding. The essential functions of BiP in facilitating proper protein folding are mainly mediated through its dynamic interaction with unfolded or misfolded client proteins, and by serving as a negative regulator of the Unfolded Protein Response. A mechanistic understanding of the dynamics of BiP interaction with its protein partners is essential to understand ER biology, and therefore, we have sought to develop a tractable model to study misfolded protein interaction with BiP. For this purpose, we have used homologous recombination to insert a 3xFLAG epitope tag into the endogenous murine Hspa5 gene, just upstream from the essential KDEL signal necessary for ER localization of BiP. Tagging BiP in this way did not alter Hspa5 expression under basal or ER-stress induced conditions in hepatocytes ex vivo or in fibroblasts. Furthermore, the tag did not alter the cellular localization of BiP or its functionality. All of these findings in primary tissue culture were also confirmed in vivo in livers of heterozygous mice harboring one WT and one FLAG-tagged Hspa5 allele. Hepatocyte-specific BiP-FLAG modification did not alter liver function or UPR signaling. Importantly, immunoprecipitation with anti-FLAG antibody completely pulled down FLAG-tagged BiP from lysates of BiP-FLAG expressing livers. Affinity purification-mass spectrometry (AP-MS) of BiP-FLAG protein complexes isolated from the BiP-FLAG-expressing livers of tunicamycin (TM)- and vehicle-treated mice revealed a marked increase in interaction of glycoproteins with BiP-FLAG in response to inhibition of N-glycosylation due to TM-treatment, validating utility of our BiP-Flag mice as a tool to identify ER misfolded proteins in vivo. Significantly, our AP-MS analysis also provided in vivo evidence demonstrating that BiP-FLAG binds to UPR transducers IRE1 and PERK under basal conditions but is released upon TM-treatment to activate UPR. We have also employed this mouse model to demonstrate that proinsulin in pancreatic {beta} cells misfolds and interacts with BiP-FLAG in healthy mice. In summary, we generated a novel model that can be used to investigate the BiP interactome in vivo under physiological and pathophysiological conditions in a cell type-specific manner. This tool provides, for the first time, an unbiased approach to identify unfolded and misfolded BiP-client proteins, and a new approach to study ER protein misfolding in a cell-type and temporal manner to uncover the role of BiP in many essential ER processes. Ear pinnae in a neotropical katydid (Orthoptera: Tettigoniidae) function as ultrasound guides for bat detection Early predator detection is a key component of the predator-prey arms race, and has driven the evolution of multiple animal hearing systems. Katydids (Insecta) have sophisticated ears, each consisting of paired tympana on each foreleg that receive sound directly externally, and internally via a narrowing ear canal through the acoustic spiracle. These ears are pressure-time difference receivers capable of sensitive and accurate directional hearing across a wide frequency range, despite the small size of katydids. Many katydid species have cuticular pinnae which form cavities around the outer tympanal surfaces, but their function is unknown. We investigated pinnal function in the katydid Copiphora gorgonensis by combining experimental biophysics and numerical modelling using 3D ear geometries. Results show that the pinnae in C. gorgonensis do not assist in directional hearing for specific call frequencies, but instead pinnae act as ultrasound detector devices. Pinnae induced large sound pressure gains that enhanced sound detection at high ultrasonic frequencies (> 60 kHz), matching the echolocation range of co-occurring insectivorous bats. Comparing pinnal mechanics of sympatric katydid species supports these findings, and suggests that pinnae evolved in katydids primarily for enhanced predator detection. Audiograms (both behavioural and neural) and tympanal cavity resonances obtained from living specimens corroborate our findings. Robust and Remote Measurement of Heart Rate Based on A Surveillance Camera Arrhythmia is a marked symptom of many cardiovascular diseases (CVDs). The accurate and timely detection of heart rate can greatly reduce the harm of arrhythmia to people. However, it is still a challenge to robustly and remotely measure heart rate in daily life due to the changing environmental conditions during measurement, such as the varying light intensity, the movement of people, and the uncertain distance between the sensor to people. In this study, we propose a method to accurately measure human heart rate within a distance of 4.5 meters under different light intensities by simply using a surveillance camera. After a 20-second color video of a persons hand is captured by the camera, a method based on the Fast Fourier Transform (FFT) algorithm is designed to extract the blood volume pulse wave to calculate the heart rate. According to the comparison between the real heart rate and results measured by electrocardiography (ECG), the proposed method achieves an accuracy of 95.8% when the measurement is performed within a distance of 4.5 meters and 90% when within 5.0 meters. Our experiments show that when the illuminance varies between 100-1000 lux (lighting level indoor), we still get the correct results. Our experiments also demonstrate that the proposed method accurately obtain heart rate even when the light intensity is below 32 lux (300-500 lux in a workplace environment). The methods strong adaptability to changing environmental conditions makes it applicable to many scenarios, such as homes of the elderly, classrooms, and other public spaces. A high throughput lipidomics method using scheduled multiple reaction monitoring Lipid compositions of cells, tissues and bio-fluids are complex, with varying concentrations and structural diversity, which makes their identification challenging. Newer methods for comprehensive analysis of lipids are thus necessary. Herein, we propose a targeted-mass spectrometry based method for large-scale lipidomics using a combination of variable retention time window and relative dwell time weightage. Using this, we detected more than 1000 lipid species, including structural isomers. The limit of detection varied from femtomolar to nanomolar range and the coefficient of variance <30% for 849 lipid species. We used this method to identify lipids altered due to Vitamin B12 deficiency and found that the levels of lipids with {omega}-3 fatty acid chains decreased while those with {omega}-6 increased. This method enables identification of by far the largest number of lipid species with structural isomers in a single experiment and would significantly advance our understanding of the role of lipids in biological processes. The evolution of mechanisms to produce phenotypic heterogeneity in microorganisms In bacteria and other microorganisms, the cells within a population often show extreme phenotypic variation. Different species use different mechanisms to determine how distinct phenotypes are allocated between individuals, including coordinated, random, and genetic determination. However, it is not clear if this diversity in mechanisms is adaptive--arising because different mechanisms are favoured in different environments--or is merely the result of non-adaptive artifacts of evolution. We use theoretical models to analyse the relative advantages of the two dominant mechanisms to divide labour between reproductives and helpers in microorganisms. We show that coordinated specialisation is more likely to evolve over random specialisation in well-mixed groups when: (i) social groups are small; (ii) helping is more "essential"; and (iii) there is a low metabolic cost to coordination. We find analogous results when we allow for spatial structure with a more detailed model of cellular filaments. More generally, this work shows how diversity in the mechanisms to produce phenotypic heterogeneity could have arisen as adaptations to different environments. High-throughput analysis of DNA replication in single human cells reveals the complex nature of replication timing control DNA replication initiates from replication origins firing throughout S phase. Debate remains about whether origins are a fixed set of loci, or a loose agglomeration of potential sites used stochastically in individual cells, and about how consistent their firing time is. We developed an approach to profile DNA replication from whole-genome sequencing of thousands of single cells, which includes "in silico flow cytometry", a method for discriminating replicating and non-replicating cells. Using two microfluidic platforms, we analyzed up to 2,437 replicating cells from a single sample. The resolution and scale of the data allow focused analysis of replication initiation sites, demonstrating that most occur in confined genomic regions. While initiation order is remarkably similar across cells, we unexpectedly identify several subtypes of initiation regions in late-replicating regions. Taken together, high throughput, high resolution sequencing of individual cells reveals previously underappreciated variability in replication initiation and progression. Citation needed?Wikipedia bibliometrics during the first waveof the COVID-19 pandemic. BackgroundWith the COVID-19 pandemics outbreak, millions flocked to Wikipedia for updated information. Amid growing concerns regarding an "infodemic", ensuring the quality of information is a crucial vector of public health. Investigating if and how Wikipedia remained up to date and in line with science is key to formulating strategies to counter misinformation. Using citation analyses, we asked: which sources informed Wikipedias COVID-19-related articles before and during the pandemics first wave (January-May 2020).

ResultsWe found that coronavirus-related articles referenced trusted media sources and high-quality academic research. Moreover, despite a surge in COVID-19 preprints, Wikipedia had a clear preference for open-access studies published in respected journals and made little use of preprints. Building a timeline of English COVID-19 articles from 2001-2020 revealed a nuanced trade-off between quality and timeliness. It further showed how preexisting articles on key topics related to the virus created a framework for integrating new knowledge. Supported by a rigid sourcing policy, this "scientific infrastructure" facilitated contextualization and regulated the influx of new information. Lastly, we constructed a network of DOI-Wikipedia articles, which showed the shifting landscape of pandemic-related knowledge on Wikipedia and how academic citations create a web of shared knowledge supporting topics like COVID-19 vaccine development.

ConclusionsUnderstanding how scientific research interacts with the digital knowledge-sphere during the pandemic provides insight into how Wikipedia can facilitate access to science. It also reveals how, aided by what we term its "citizen encyclopedists", it successfully fended off COVID-19 disinformation and how this unique model may be deployed in other contexts. Deep Heterogeneous Dilation of LSTM for Transient-phase Gesture Prediction through High-density Electromyography: Towards Application in Neurorobotics Deep networks have been recently proposed to estimate motor intention using conventional bipolar surface electromyography (sEMG) signals for myoelectric control of neurorobots. In this regard, Deepnets are generally challenged by long training times (affecting practicality and calibration), complex model architectures (affecting the predictability of the outcomes), and a large number of trainable parameters (increasing the need for big data). Capitalizing on our recent work on homogeneous temporal dilation in a Recurrent Neural Network (RNN) model, this paper proposes, for the first time, heterogeneous temporal dilation in an LSTM model and applies that to high-density surface electromyography (HD-sEMG), allowing for the decoding of dynamic temporal dependencies with tunable temporal foci. In this paper, a 128-channel HD-sEMG signal space is considered due to the potential for enhancing the spatiotemporal resolution of human-robot interfaces. Accordingly, this paper addresses a challenging motor intention decoding problem of neurorobots, namely, transient intention identification. Our approach uses only the dynamic and transient phase of gesture movements when the signals are not stabilized or plateaued, which can significantly enhance the temporal resolution of human-robot interfaces. This would eventually enhance seamless real-time implementations. Additionally, this paper introduces the concept of "dilation foci" to modulate the modeling of temporal variation in transient phases. In this work a high number (e.g., 65) of gestures is included, which adds to the complexity and significance of the understudied problem. Our results show state-of-the-art performance for gesture prediction in terms of accuracy, training time, and model convergence. An integrative approach leads to the discovery of a novel anti-leukemic peptide from human milk Chemotherapy in childhood leukemia is associated with late morbidity in leukemic survivors, while certain patient subsets are relatively resistant to standard chemotherapy. It is therefore important to identify new agents with sensitivity and selectivity towards leukemic cells, while having less systemic toxicity. Peptide-based therapeutics has gained much attention during the last few years. Here, we used an integrative workflow combining mass spectrometric peptide library construction, in silico anticancer peptide screening, and in vitro leukemic cell studies to discover a novel anti-leukemic peptide having 3+charges and alpha-helical structure, namely HMP-S7, from human breast milk. HMP-S7 showed cytotoxic activity against four distinct leukemic cell lines in a dose-dependent manner but had no effect on solid malignancies or representative normal cells. HMP-S7 induced leukemic cell death by penetrating the plasma membrane to enter the cytoplasm and cause leakage of lactate dehydrogenase, thus acting in a membranolytic manner. Importantly, HMP-S7 exhibited anti-leukemic effect against patient-derived leukemic cells ex vivo. In conclusion, HMP-S7 is a selective anti-leukemic peptide with promise which requires further validation in preclinical and clinical studies.

TeaserIn silico screening of naturally occurring human milk peptides discovers a new anticancer peptide that kills leukemic cells in vitro and ex vivo. Changes in Behavior and Salivary Serum Amyloid A in cows with Subclinical Mastitis The aim of this study was to identify detailed changes in behavior, and in salivary serum amyloid A (SAA), associated with subclinical mastitis. This included standard sickness behaviors (which form part of an adaptive response to conserve energy, minimize heat loss and facilitate recovery following infection and inflammation) and lesser-studied behaviors, that are biologically predicted to change at low-level infection, and therefore particularly relevant for detecting subclinical mastitis (here labelled  luxury behaviors). SAA is a physiological marker of systemic inflammation, with levels in milk and serum already known to increase during subclinical mastitis. We investigated whether the same was true for SAA in cow saliva. Data were collected for n = 34 commercial barn-housed dairy cows, tested in 17 matched-pairs. Each pair comprised a cow with subclinical mastitis (SCM) and a healthy control (CTRL), identified using somatic cell count (SCC) (SCM: SCC >200 x1000 cells/ml; CTRL: SCC <100 x1000 cells/ml). SCM cows were selected for study ad-hoc, at which point they were paired with a CTRL cow, based upon parity and calving date; consequently, the full data set was accrued over several months. Data were collected for each pair over 3 days: SCC (l4:00-l5:00h) Day 1; behavior (24h from 00:00h) Day 2; salivary serum amyloid-A (SAA) Day 3. We report, for the first time, that an increase in salivary SAA occurs during subclinical mastitis; SAA was higher in SCM cows and demonstrated a positive (weak) correlation with SCC. The behavioral comparisons revealed that SCM cows had reductions in activity (behavioral transitions and distance moved), social exploration, social reactivity (here: likelihood to move away/be displaced following receipt of agonism), performance of social grooming and head butts, and the receipt of agonistic non-contact challenges. In addition, SCM cows received more head swipes, and spent a greater proportion of time lying with their head on their flank than CTRL cows. SCM cows also displayed an altered feeding pattern; they spent a greater proportion of feeding time in direct contact with two conspecifics, and a lower proportion of feeding time at self-locking feed barriers, than CTRL cows. Behavioral measures were found to correlate, albeit loosely, with serum SAA in a direction consistent with predictions for sickness behavior. These included positive correlations with both lying duration and the receipt of all agonistic behavior, and negative correlations with feeding, drinking, the performance of all social and all agonistic behavior, and social reactivity. We conclude that changes in salivary SAA, social behavior, and activity offer potential in the detection of subclinical mastitis and recommend further investigation to substantiate and refine our findings. Fatal COVID-19 and non-COVID-19 Acute Respiratory Distress Syndrome is Associated with Incomplete Alveolar Type 1 Epithelial Cell Differentiation from the Transitional State Without Fibrosis ARDS due to COVID-19 and other etiologies results from injury to the alveolar epithelial cell (AEC) barrier resulting in noncardiogenic pulmonary edema, which causes acute respiratory failure; clinical recovery requires epithelial regeneration. During physiologic regeneration in mice, AEC2s proliferate, exit the cell cycle, and transiently assume a transitional state before differentiating into AEC1s; persistence of the transitional state is associated with pulmonary fibrosis in humans. It is unknown whether transitional cells emerge and differentiate into AEC1s without fibrosis in human ARDS and why transitional cells differentiate into AEC1s during physiologic regeneration but persist in fibrosis. We hypothesized that incomplete but ongoing AEC1 differentiation from transitional cells without fibrosis may underlie persistent barrier permeability and fatal acute respiratory failure in ARDS. Immunostaining of postmortem ARDS lungs revealed abundant transitional cells in organized monolayers on alveolar septa without fibrosis. They were typically cuboidal or partially spread, sometimes flat, and occasionally expressed AEC1 markers. Immunostaining and/or interrogation of scRNAseq datasets revealed that transitional cells in mouse models of physiologic regeneration, ARDS, and fibrosis express markers of cell cycle exit but only in fibrosis express a specific senescence marker. Thus, in severe, fatal early ARDS, AEC1 differentiation from transitional cells is incomplete, underlying persistent barrier permeability and respiratory failure, but ongoing without fibrosis; senescence of transitional cells may be associated with pulmonary fibrosis. Diverse stem-chondrichthyan oral structures and evidence for an independently acquired acanthodid dentition The teeth of sharks famously form a series of transversely-organised files with conveyor-belt replacement that are borne directly on the jaw cartilages, in contrast to the dermal plate-borne dentition of bony fishes that undergoes site-specific replacement. A major obstacle in understanding how this system evolved is the poorly understood relationships of the earliest chondrichthyans and the profusion of morphologically and terminologically diverse bones, cartilages, splints and whorls that they possess. Here we use tomographic methods to investigate mandibular structures in several early branching  acanthodian-grade stem-chondrichthyans. We show that the dentigerous jaw bones of disparate genera of ischnacanthids are united by a common construction, being growing bones with non-shedding dentition. Mandibular splints, which support the ventro-lateral edge of the Meckels cartilage in some taxa, are formed from dermal bone and may be an acanthodid synapomorphy. We demonstrate that the teeth of Acanthodopsis are borne directly on the mandibular cartilage and that this taxon is deeply nested within an edentulous radiation, representing an unexpected independent origin of teeth. Many or even all of the range of unusual oral structures may be apomorphic, but they should nonetheless be considered when building hypotheses of tooth and jaw evolution, both in chondrichthyans and more broadly. A natural mutator allele shapes mutation spectrum variation in mice Summary ParagraphAlthough germline mutation rates and spectra can vary within and between species, genetic modifiers of these traits have long eluded detection. In this study, we searched for loci that influence germline mutagenesis using a uniquely powerful resource: a panel of recombinant inbred mouse lines known as the BXD, descended from the laboratory mouse strains C57BL/6J (B) and DBA/2J (D). Each BXD lineage has been maintained by brother-sister mating in the near absence of natural selection, accumulating de novo mutations for up to 50 years on a known genetic background that is a unique linear mosaic of B and D haplotypes. We show that mice inheriting D haplotypes at a quantitative trait locus (QTL) on chromosome 4 accumulate C>A germline mutations at a 50% higher rate than those inheriting B haplotypes, primarily due to the activity of a C>A-dominated mutational signature known as SBS18. The B and D QTL haplotypes encode different alleles of the DNA repair gene Mutyh, which underlies the heritable colorectal cancer syndrome in which SBS18 was first identified. The B and D Mutyh alleles are present in wild populations of Mus musculus domesticus, providing evidence that common genetic variation modulates germline mutagenesis in a model mammalian species. Towards a unified model of naive T cell dynamics across the lifespan Naive CD4 and CD8 T cells are cornerstones of adaptive immunity, but the dynamics of their establishment early in life and how their kinetics change as they mature following release from the thymus are poorly understood. Further, due to the diverse signals implicated in naive T cell survival, it has been a long-held and conceptually attractive view that they are sustained by active homeostatic control as thymic activity wanes. Here we employ multiple experimental systems to identify a unified model of naive CD4 and CD8 T cell population dynamics across the mouse lifespan. We infer that both subsets divide rarely and progressively increase their survival capacity with cell age. Strikingly, this simple model captures naive CD4 T cell dynamics throughout life. In contrast, we find that newly generated naive CD8 T cells are lost more rapidly during the first 3-4 weeks of life, likely due to increased recruitment into memory. We find no evidence for elevated division rates in neonates, or for feedback regulation of naive T cell numbers at any age. We show how confronting mathematical models with diverse datasets can reveal a quantitative and remarkably simple picture of naive T cell dynamics from birth into old age. RNAi screen of RING/U-box domain ubiquitin ligases identifies critical regulators of tissue regeneration in planarians Regenerative processes depend on the interpretation of signals to coordinate cell behaviors. The role of ubiquitin-mediated signaling is known to be important in many cellular and biological contexts, but its role in regeneration is not well understood. To investigate how ubiquitylation impacts tissue regeneration in vivo, we are studying planarians that are capable of regenerating after nearly any injury using a population of stem cells. Here we used RNAi to screen RING/U-box E3 ubiquitin ligases that are highly expressed in planarian stem cells and stem cell progeny. RNAi screening identified nine genes with functions in regeneration, including the spliceosomal factor prpf19 and histone modifier rnf2; based on their known roles in developmental processes, we further investigated these two genes. We found that prpf19 was required for animal survival but not for stem cell maintenance, suggesting a role in promoting cell differentiation. Because RNF2 is the catalytic subunit of the Polycomb Repressive Complex 1 (PRC1), we also examined other putative members of this complex (CBX and PHC). We observed a striking phenotype of regional tissue misspecification in cbx and phc RNAi planarians. To identify genes regulated by PRC1, we performed RNA-seq after knocking down rnf2 or phc. Although these proteins are predicted to function in the same complex, we found that the set of genes differentially expressed in rnf2 versus phc RNAi were largely non-overlapping. Using in situ hybridization, we showed that rnf2 regulates gene expression levels within a tissue type, whereas phc is necessary for the spatial restriction of gene expression, findings consistent with their respective in vivo phenotypes. This work not only uncovered roles for RING/U-box E3 ligases in stem cell regulation and regeneration, but also identified differential gene targets for two putative PRC1 factors required for maintaining cell-type-specific gene expression in planarians. Unevolved proteins from modern and prebiotic amino acids manifest distinct structural profiles Natural proteins represent numerous but tiny structure/function islands in a vast ocean of possible protein sequences, most of which has not been explored by either biological evolution or research. Recent studies have suggested this uncharted sequence space possesses surprisingly high structural propensity, but development of an understanding of this phenomenon has been awaiting a systematic high-throughput approach.

Here, we designed, prepared, and characterized two combinatorial protein libraries consisting of randomized proteins, each 105 residues in length. The first library constructed proteins from the entire canonical alphabet of 20 amino acids. The second library used a subset of only 10 residues (A,S,D,G,L,I,P,T,E,V) that represent a consensus view of plausibly available amino acids through prebiotic chemistry. Our study shows that compact conformations resistant to proteolysis are (i) abundant (up to 40%) in random sequence space, (ii) independent of general Hsp70 chaperone system activity, and (iii) not granted solely by "late" and complex amino acid additions. The Hsp70 chaperone system effectively increases solubility and refoldability of the canonical alphabet but has only a minor impact on the "early" library. The early alphabet proteins are inherently more soluble and refoldable, possibly assisted by the cell-like environment in which these assays were performed.

Our work indicates that both early and modern amino acids are predisposed to supporting protein structure (either in forms of oligomers or globular/molten globule structures) and that protein structure may not be a unique outcome of evolution. Emergent dynamics of a three-node regulatory network explain phenotypic switching and heterogeneity: a case study of Th1/Th2/Th17 cell differentiation Naive helper (CD4+) T-cells can differentiate into distinct functional subsets including Th1, Th2, and Th17 phenotypes. Each of these phenotypes has a  master regulator - T-bet (Th1), GATA3 (Th2) and ROR{gamma}T (Th17) - that inhibits the other two master regulators. Such mutual repression among them at a transcriptional level can enable multistability, giving rise to six experimentally observed phenotypes - Th1, Th2, Th17, hybrid Th/Th2, hybrid Th2/Th17 and hybrid Th1/Th17. However, the dynamics of switching among these phenotypes, particularly in the case of epigenetic influence, remains unclear. Here, through mathematical modeling, we investigated the coupled transcription-epigenetic dynamics in a three-node mutually repressing network to elucidate how epigenetic changes mediated by any  master regulator can influence the transition rates among different cellular phenotypes. We show that the degree of plasticity exhibited by one phenotype depends on relative strength and duration of mutual epigenetic repression mediated among the master regulators in a three-node network. Further, our model predictions can offer putative mechanisms underlying relatively higher plasticity of Th17 phenotype as observed in vitro and in vivo. Together, our modeling framework characterizes phenotypic plasticity and heterogeneity as an outcome of emergent dynamics of a three-node regulatory network, such as the one mediated by T-bet/GATA3/ROR{gamma}T. Temperature Increase Drives Critical Slowing Down of Fish Ecosystems Fish ecosystems perform ecological functions that are critically important for the sustainability of marine ecosystems, such as global food security and carbon stock. During the 21st century, significant global warming caused by climate change has created pressing challenges for fish ecosystems that threaten species existence and global ecosystem health. Here, we study a coastal fish community in Maizuru Bay, Japan, and investigate the relationships between fluctuations of ST, abundance-based species interactions and salient fish biodiversity. Observations show that a local 20% increase in temperature from 2002 to 2014 underpins a long-term reduction in fish diversity ([~]25%) played out by some native and invasive species (e.g. Chinese wrasse) becoming exceedingly abundant; this causes a large decay in commercially valuable species (e.g. Japanese anchovy) coupled to an increase in ecological productivity. The fish community is analyzed considering five temperature ranges to understand its atemporal seasonal sensitivity to ST changes, and long-term trends. An optimal information flow model is used to reconstruct species interaction networks that emerge as topologically different for distinct temperature ranges and species dynamics. Networks for low temperatures are more scale-free compared to ones for intermediate (15-20{degrees}C) temperatures in which the fish ecosystem experiences a first-order phase transition in interactions from locally stable to metastable and globally unstable for high temperatures states as suggested by abundance-spectrum transitions. The dynamic dominant eigenvalue of species interactions shows increasing instability for competitive species (spiking in summer due to intermediate-season critical transitions) leading to enhanced community variability and critical slowing down despite higher time-point resilience. Native competitive species whose abundance is distributed more exponentially have the highest total directed interactions and are keystone species (e.g. Wrasse and Horse mackerel) for the most salient links with cooperative decaying species. Competitive species, with higher eco-climatic memory and synchronization, are the most affected by temperature and play an important role in maintaining fish ecosystem stability via multitrophic cascades (via cooperative-competitive species imbalance), and as bioindicators of change. More climate-fitted species follow temperature increase causing larger divergence divergence between competitive and cooperative species. Decreasing dominant eigenvalues and lower relative network optimality for warmer oceans indicate fishery more attracted toward persistent oscillatory states, yet unpredictable, with lower cooperation, diversity and fish stock despite the increase in community abundance due to non-commercial and venomous species. We emphasize how changes in species interaction organization, primarily affected by temperature fluctuations, are the backbone of biodiversity dynamics and yet for functional diversity in contrast to taxonomic richness. Abundance and richness manifest gradual shifts while interactions show sudden shift. The work provides data-driven tools for analyzing and monitoring fish ecosystems under the pressure of global warming or other stressors. Abundance and interaction patterns derived by network-based analyses proved useful to assess ecosystem susceptibility and effective change, and formulate predictive dynamic information for science-based fishery policy aimed to maintain marine ecosystems stable and sustainable. Phytoene synthase 2 in tomato fruits remains functional and contributes to abscisic acid formation In ripening tomato fruits, the leaf-specific carotenoids biosynthesis mediated by phytoene synthase 2 (PSY2) is replaced by a fruit-specific pathway by the expression of two chromoplast-specific genes: phytoene synthase 1 (PSY1) and lycopene-{beta}-cyclase (CYCB). Though both PSY1 and PSY2 genes express in tomato fruits, the functional role of PSY2 is not known. To decipher whether PSY2-mediated carotenogenesis operates in ripening fruits, we blocked the in vivo activity of lycopene-{beta}-cyclases in fruits of several carotenoids and ripening mutants by CPTA (2-(4-Chlorophenylthio)triethylamine hydrochloride), an inhibitor of lycopene-{beta}-cyclases. The CPTA-treatment induced accumulation of lycopene in leaves, immature-green and ripening fruits. Even, in psy1 mutants V7 and r that are deficient in fruit-specific carotenoid biosynthesis, CPTA triggered lycopene accumulation but lowered the abscisic acid level. Differing from fruit-specific carotenogenesis, CPTA-treated V7 and r mutant fruits accumulated lycopene but not phytoene and phytofluene. The lack of phytoene and phytofluene accumulation was reminiscent of PSY2-mediated leaf-like carotenogenesis, where phytoene and phytofluene accumulation is never seen. The lycopene accumulation was associated with the partial transformation of chloroplasts to chromoplasts bearing thread-like crystalline structures. Our study uncovers the operation of a parallel carotenogenesis pathway mediated by PSY2 that provides precursors for abscisic acid biosynthesis in ripening tomato fruits.

Significance statementIt is believed that in ripening tomato fruits phytoene synthase 2 that drives carotenoid biosynthesis in leaves is redundant. Contrary to this, we show that in phytoene synthase 1 mutant fruit that is bereft of lycopene, the chemical inhibition of lycopene {beta}-cyclases triggers lycopene accumulation. Our results uncover that phytoene synthase 2 remains functional in ripening fruits and provides precursors for abscisic acid formation in fruits.