bluuebunny/crossref_metadata_2025 huggingface dataset:
Magnetic Resonance Imaging for Evaluation of Interstitial Fibrosis in Kidney Allografts
Authors: Beck-Tölly Andrea , Eder Michael , Beitzke Dietrich , Eskandary Farsad , Agibetov Asan , Lampichler Katharina , Hamböck Martina , Regele Heinz , Kläger Johannes , Nackenhorst Maja , Böhmig Georg A.
Published: December 2020

Background.

Interstitial fibrosis (IF) is the common pathway of chronic kidney injury in various conditions. Magnetic resonance imaging (MRI) may be a promising tool for the noninvasive assessment of IF in renal allografts.

Methods.

This prospective trial was primarily designed to investigate whether the results of T1-weighted MRI associate with the degree of IF. Thirty-two kidney transplant recipients were subjected to 1.5-Tesla MRI scans shortly before or after routine allograft biopsies. MRI parameters [T1 and T2 relaxation times; apparent diffusion coefficient (ADC)] were assessed for cortical and medullary sections.

Results.

Advanced IF (Banff ci score >1) was associated with higher cortical T1 (but not T2) values [1451 (median; interquartile range: 1331–1506) versus 1306 (1197–1321) ms in subjects with ci scores ≤1; P = 0.011; receiver operating characteristic area under the curve for prediction of ci > 1: 0.76]. In parallel, T1 values were associated with kidney function and proteinuria. There was also a relationship between IF and corticomedullary differences on ADC maps (receiver operating characteristic area under the curve for prediction of ci ≤ 1: 0.79).

Conclusions.

Our results support the use of MRI for noninvasive assessment of allograft scarring. Future studies will have to clarify the role of T1 (and ADC) mapping as a surrogate endpoint reflecting the progression of chronic graft damage.
COVID-19 in Kidney Transplantation: Epidemiology, Management Considerations, and the Impact on Kidney Transplant Practice
Authors: Kataria Ashish , Yakubu Idris , Winstead Ryan , Gowda Madan , Gupta Gaurav
Published: December 2020

The novel severe acute respiratory syndrome coronavirus 2 was identified in the late 2019 as the cause of coronavirus disease 2019 (COVID-19), an acute respiratory viral illness. Patients with chronic underlying conditions may have an increased risk of morbidity and mortality from COVID-19. Kidney transplant recipients may be at a uniquely increased risk of serious complications from COVID-19 as compared to the general population because of a chronically immunosuppressed state and a high prevalence of comorbidities like diabetes, heart disease, and lung disease. Early data suggest that the mortality of patients on dialysis may be comparable to those with kidney transplants, although more research is needed. This concise review aims to describe the epidemiology of COVID-19 in kidney transplant recipients, manifestations, appropriate management, and clinical outcomes based on the available literature. Current evidence on many of the specific antiviral measures against COVID-19 has not shown a clear-cut benefit in smaller studies and the results of several ongoing larger clinical trials are awaited. In addition, we also highlight the impact of COVID-19 on kidney transplant center practice and volumes; potential living or deceased donors, recipients; and induction immunosuppression and surgical strategies.
Significant Improvement in Rat Kidney Cold Storage Using UW Organ Preservation Solution Supplemented With the Immediate-Acting PrC-210 Free Radical Scavenger
Authors: Verhoven Bret M. , Karim Aos S. , Bath Natalie M. , Sarabia Fahl Carol J. , Wilson Nancy A. , Redfield Robert R. , Fahl William E.
Published: December 2020

Background.

Ischemia-reperfusion injury, including injury from warm- and cold-ischemia (CI) organ storage, remains a significant problem for all solid organ transplants. Suppressing CI damage would reduce delayed graft function and increase the donor organ pool size. PrC-210 has demonstrated superior prevention of damage in several preclinical studies as an immediate-acting free-radical scavenger. Here, we describe its profound efficacy in suppressing CI injury in a rat kidney model.

Methods.

Kidneys in 300 gm Sprague-Dawley rats were perfused in situ with UW solution with or without added PrC-210 and then stored at 4°C in the same solution for 0 to 48 hours. When procured, kidney-activated caspase-3 level (a marker of cell death) was measured, and direct histological analysis of kidneys was performed to assess PrC-210 protective efficacy. In vitro analyses of PrC-210-conferred protection to isolated rat kidneys or naked DNA were also performed.

Results.

A single 15 seconds in situ perfusion of kidneys with 20 mmol/L PrC-210 in UW solution resulted in significant reductions in (1) 30-hour CI–induced kidney-activated caspase level (P < 0.0001); activated caspase was reduced to levels not significantly different than control activated caspase levels seen in unperturbed kidneys, (2) 30-hour CI–induced renal Tubular Injury Scores (P = 0.0004) where brush border and tubular necrosis were markedly reduced, (3) PrC-210 conferred 100% protection against ·OH damage to naked DNA and isolated kidney mitochondria while current UW solution antioxidants were without protective effect.

Conclusions.

A single PrC-210-UW solution perfusion of rat kidneys upon removal from the rat profoundly reduced caspase and renal tubular injury in kidneys exposed to 30 hours of CI organ storage. These findings support further development of the PrC-210 molecule to suppress or to prevent ischemia-reperfusion injury in organ transplant and other ischemia-reperfusion injury settings.
Evidence for GTKO/β4GalNT2KO Pigs as the Preferred Organ-source for Old World Nonhuman Primates as a Preclinical Model of Xenotransplantation
Authors: Cui Yehua , Yamamoto Takayuki , Raza Syed Sikandar , Morsi Mahmoud , Nguyen Huy Quoc , Ayares David , Cooper David K.C. , Hara Hidetaka
Published: December 2020

Background.

Triple-knockout (TKO) pigs (in which expression of the 3 known pig carbohydrate xenoantigens has been deleted) are likely to be an optimal source of organs for transplantation into human recipients, many of whom do not have natural antibodies against TKO pig cells. However, old world monkeys, for example, baboons, have natural antibodies directed to TKO cells (to a “fourth” xenoantigen that is exposed after TKO).

Methods.

We measured (1) anti-pig IgM/IgG binding, and (2) complement-dependent cytotoxicity (CDC), by flow cytometry to α1,3-galactosyltransfearse gene-knockout (GTKO), GTKO/β4GalNT2KO (that do not express the “fourth” xenoantigen), and TKO pig peripheral blood mononuclear cells (PBMCs) using 72 baboon sera (30 specific pathogen-free [SPF], and 42 non-SPF baboons).

Results.

Mean IgM antibody binding to GTKO/β4GalNT2KO pig PBMCs was significantly lower than to GTKO or TKO pig PBMCs (P < 0.01). Mean IgG antibody binding to GTKO/β4GalNT2KO pig PBMCs was significantly lower than to TKO PBMCs (P < 0.01). Mean CDC of GTKO/β4GalNT2KO pig PBMCs was significantly lower than of GTKO or TKO pig PBMCs (P < 0.01). SPF baboon serum IgM and IgG binding to, and CDC of, GTKO/β4GalNT2KO or TKO PBMCs were significantly lower than non-SPF baboon sera (P < 0.01).

Conclusions.

Although TKO pigs form the basis for proposed clinical trials of xenotransplantation, it is difficult to identify baboons with a low or negative CDC to TKO pigs. For pig-to-baboon organ transplantation, the use of GTKO/β4GalNT2KO pigs would be preferable. The use of SPF baboons as recipients might be a minor advantage.
Diffusion in arrays of obstacles: beyond homogenization
Authors: Farah Yahya , Loghin Daniel , Tzella Alexandra , Vanneste Jacques
Published: December 2020

We revisit the classical problem of diffusion of a scalar (or heat) released in a two-dimensional medium with an embedded periodic array of impermeable obstacles such as perforations. Homogenization theory provides a coarse-grained description of the scalar at large times and predicts that it diffuses with a certain effective diffusivity, so the concentration is approximately Gaussian. We improve on this by developing a large-deviation approximation which also captures the non-Gaussian tails of the concentration through a rate function obtained by solving a family of eigenvalue problems. We focus on cylindrical obstacles and on the dense limit, when the obstacles occupy a large area fraction and non-Gaussianity is most marked. We derive an asymptotic approximation for the rate function in this limit, valid uniformly over a wide range of distances. We use finite-element implementations to solve the eigenvalue problems yielding the rate function for arbitrary obstacle area fractions and an elliptic boundary-value problem arising in the asymptotics calculation. Comparison between numerical results and asymptotic predictions confirms the validity of the latter.
A statistical theory of the strength of epidemics: an application to the Italian COVID-19 case
Authors: Pisano Gabriele , Royer-Carfagni Gianni
Published: December 2020

The proposed theory defines a relative index of epidemic lethality that compares any two configurations in different observation periods, preferably one in the acute and the other in a mild epidemic phase. Raw mortality data represent the input, with no need to recognize the cause of death. Data are categorized according to the victims’ age, which must be renormalized because older people have a greater probability of developing a level of physical decay (human damage), favouring critical pathologies and co-morbidities. The probabilistic dependence of human damage on renormalized age is related to a death criterion considering a virus spread by contagion and our capacity to cure the disease. Remarkably, this is reminiscent of the Weibull theory of the strength of brittle structures containing a population of crack-like defects, in the correlation between the statistical distribution of cracks and the risk of fracture at a prescribed stress level. Age-of-death scaling laws are predicted in accordance with data collected in Italian regions and provinces during the first wave of COVID-19, taken as representative examples to validate the theory. For the prevention of spread and the management of the epidemic, the various parameters of the theory shall be informed on other existing epidemiological models.
Capillary transport in paper porous materials at low saturation levels: normal, fast or superfast?
Authors: Lukyanov Alex V. , Mitkin Vladimir V. , Pryer Tristan , Sirimark Penpark , Theofanous Theo G.
Published: December 2020

The problem of capillary transport in fibrous porous materials at low levels of liquid saturation has been addressed. It has been demonstrated that the process of liquid spreading in this type of porous material at low saturation can be described macroscopically by a similar super-fast, nonlinear diffusion model to that which had been previously identified in experiments and simulations in particulate porous media. The macroscopic diffusion model has been underpinned by simulations using a microscopic network model. The theoretical results have been qualitatively compared with available experimental observations within the witness card technique using persistent liquids. The long-term evolution of the wetting spots was found to be truly universal and fully in line with the mathematical model developed. The result has important repercussions for the witness card technique used in field measurements of the dissemination of various low-volatility agents in imposing severe restrictions on collection and measurement times.
Mercury exposure in an endangered seabird: long-term changes and relationships with trophic ecology and breeding success
Authors: Mills William F. , Bustamante Paco , McGill Rona A. R. , Anderson Orea R. J. , Bearhop Stuart , Cherel Yves , Votier Stephen C. , Phillips Richard A.
Published: December 2020

Mercury (Hg) is an environmental contaminant which, at high concentrations, can negatively influence avian physiology and demography. Albatrosses (Diomedeidae) have higher Hg burdens than all other avian families. Here, we measure total Hg (THg) concentrations of body feathers from adult grey-headed albatrosses (
Thalassarche chrysostoma
) at South Georgia. Specifically, we (i) analyse temporal trends at South Georgia (1989–2013) and make comparisons with other breeding populations; (ii) identify factors driving variation in THg concentrations and (iii) examine relationships with breeding success. Mean ± s.d. feather THg concentrations were 13.0 ± 8.0 µg g
−1
dw, which represents a threefold increase over the past 25 years at South Georgia and is the highest recorded in the
Thalassarche
genus. Foraging habitat, inferred from stable isotope ratios of carbon (
δ
13
C), significantly influenced THg concentrations—feathers moulted in Antarctic waters had far lower THg concentrations than those moulted in subantarctic or subtropical waters. THg concentrations also increased with trophic level (
δ
15
N), reflecting the biomagnification process. There was limited support for the influence of sex, age and previous breeding outcome on feather THg concentrations. However, in males, Hg exposure was correlated with breeding outcome—failed birds had significantly higher feather THg concentrations than successful birds. These results provide key insights into the drivers and consequences of Hg exposure in this globally important albatross population.
Population studies of the wild tomato species
Solanum chilense
reveal geographically structured major gene-mediated pathogen resistance
Authors: Kahlon Parvinderdeep S. , Seta Shallet Mindih , Zander Gesche , Scheikl Daniela , Hückelhoven Ralph , Joosten Matthieu H. A. J. , Stam Remco
Published: December 2020

Natural plant populations encounter strong pathogen pressure and defence-associated genes are known to be under selection dependent on the pressure by the pathogens. Here, we use populations of the wild tomato
Solanum chilense
to investigate natural resistance against
Cladosporium fulvum
, a well-known ascomycete pathogen of domesticated tomatoes. Host populations used are from distinct geographical origins and share a defined evolutionary history. We show that distinct populations of
S. chilense
differ in resistance against the pathogen. Screening for major resistance gene-mediated pathogen recognition throughout the whole species showed clear geographical differences between populations and complete loss of pathogen recognition in the south of the species range. In addition, we observed high complexity in a
homologues of Cladosporium resistance
(
Hcr
) locus, underlying the recognition of
C. fulvum,
in central and northern populations. Our findings show that major gene-mediated recognition specificity is diverse in a natural plant-pathosystem. We place major gene resistance in a geographical context that also defined the evolutionary history of that species. Data suggest that the underlying loci are more complex than previously anticipated, with small-scale gene recombination being possibly responsible for maintaining balanced polymorphisms in the populations that experience pathogen pressure.
Intraspecies plasmid and genomic variation of Mycobacterium kubicae revealed by the complete genome sequences of two clinical isolates
Authors: Hendrix Jo , Epperson L. Elaine , Durbin David , Honda Jennifer R. , Strong Michael
Published: December 2020

Mycobacterium kubicae

is 1 of nearly 200 species of nontuberculous mycobacteria (NTM), environmental micro-organisms that in some situations can infect humans and cause severe lung, skin and soft tissue infections. Although numerous studies have investigated the genetic variation among prevalent clinical NTM species, including

Mycobacterium abscessus

and

Mycobacterium avium

, many of the less common but clinically relevant NTM species, including

M. kubicae

, still lack complete genomes to serve as a comparative reference. Well-characterized representative genomes for each NTM species are important both for investigating the pathogenic potential of NTM, as well as for use in diagnostic methods, even for species that less frequently cause human disease. Here, we report the complete genomes of two

M. kubicae

strains, isolated from two unrelated patients. Hybrid short-read and long-read sequencing and assembly, using sequence reads from Illumina and Oxford Nanopore Technologies platforms, were utilized to resolve the chromosome and plasmid sequences of each isolate. The genome of NJH_MKUB1 had 5135 coding sequences (CDSs), a circular chromosome of length 5.3 Mb and two plasmids. The genome of NJH_MKUB2 had 5957 CDSs, a circular chromosome of 6.0 Mb and five plasmids. We compared our completed genomic assemblies to four recently released draft genomes of

M. kubicae

in order to better understand intraspecies genomic conservation and variability. We also identified genes implicated in drug resistance, virulence and persistence in the

M. kubicae

chromosome and plasmids. Virulence factors encoded in the genome and in the plasmids of

M. kubicae

provide a foundation for investigating how opportunistic environmental NTM may cause disease.
Airborne Transmission of Virus-Laden Aerosols inside a Music Classroom: Effects of Portable Purifiers and Aerosol Injection Rates
Authors: Narayanan Sai Ranjeet , Yang Suo
Published: December 2020

The ongoing COVID-19 pandemic has shifted attention to the airborne transmission of exhaled droplet nuclei within indoor environments. The spread of aerosols through singing and musical instruments in music performances has necessitated precautionary methods such as masks and portable purifiers. This study investigates the effects of placing portable air purifiers at different locations inside a classroom, as well as the effects of different aerosol injection rates (e.g., with and without masks, different musical instruments and different injection modes). Aerosol deposition, airborne concentration and removal are analyzed in this study. It was found that using purifiers could help in achieving ventilation rates close to the prescribed values by the World Health Organization (WHO), while also achieving aerosol removal times within the Center of Disease Control and Prevention (CDC) recommended guidelines. This could help in deciding break periods between classroom sessions, which was around 25 minutes through this study. Moreover, proper placement of purifiers could offer significant advantages in reducing airborne aerosol numbers (offering orders of magnitude higher aerosol removal when compared to nearly zero removal when having no purifiers), and improper placement of the purifiers could worsen the situation. The study suggests the purifier to be placed close to the injector to yield a benefit, and away from the people to be protected. The injection rate was found to have an almost linear correlation with the average airborne aerosol suspension rate and deposition rate, which could be used to predict the trends for scenarios with other injection rates.
A New Extension of State-Space SIR Model to Account for Underreporting- An Application to the COVID-19 transmission in California and Florida
Authors: Deo Vishal , Grover Gurprit
Published: December 2020

In the absence of sufficient testing capacity for COVID-19, a substantial number of infecteds are expected to remain undetected. Since the undetected cases are not quarantined, they are expected to transmit the infection at a much higher rate than their quarantined counterparts. That is, under the lack of extensive random testing, the actual prevalence and incidence of the SARS-CoV-2 infection may be entirely different from that being reported. Thus, it is imperative that the information on the percentage of undetected (or unreported) cases be considered while estimating the parameters and forecasting the transmission dynamics of the epidemic.

In this paper, we have developed a new version of the basic susceptible-infected-removed (SIR) compartmental model, called the susceptible-infected (quarantined/ free) -recovered-deceased [SI(Q/F)RD] model, to incorporate the impact of undetected cases on the transmission dynamics of the epidemic. Further, we have presented a Dirichlet-Beta state-space formulation of the SI(Q/F)RD model for the estimation of its parameters using posterior realizations from Gibbs sampling procedure. As a demonstration, the proposed methodology is implemented to forecast the COVID-19 transmission in California and Florida.

Highlights

Data calibrated for underreporting using excess deaths and case fatality rate.

A new extension of SIR compartmental model, called SI(Q/F)RD, is introduced.

A Dirichlet-Beta state-space formulation of the SI(Q/F)RD model is developed.

Gibbs sampling used to estimate the Bayesian hierarchical state-space model.

Proposed methodology is applied on the COVID-19 data of California and Florida.
Analysis of 46,046 SARS-CoV-2 whole-genomes leveraging principal component analysis (PCA)
Authors: Scherer Christiane , Grover James , Kammeraad Darby , Rudy Gabe , Scherer Andreas
Published: December 2020

Since the beginning of the global SARS-CoV-2 pandemic, there have been a number of efforts to understand the mutations and clusters of genetic lines of the SARS-CoV-2 virus. Until now, phylogenetic analysis methods have been used for this purpose. Here we show that Principal Component Analysis (PCA), which is widely used in population genetics, can not only help us to understand existing findings about the mutation processes of the virus, but can also provide even deeper insights into these processes while being less sensitive to sequencing gaps. Here we describe a comprehensive analysis of a 46,046 SARS-CoV-2 genome sequence dataset downloaded from the GISAID database in June of this year.

PCA provides deep insights into the analysis of large data sets of SARS-CoV-2 genomes, revealing virus lineages that have thus far been unnoticed.
Functional diversity of isoprenoidal lipids inMethylobacterium extorquensPA1
Authors: Rizk Sandra , Henke Petra , Santana-Molina Carlos , Martens Gesa , Gnädig Marén , Devos Damien P , Neumann-Schaal Meina , Saenz James P
Published: December 2020

Hopanoids and carotenoids are two of the major isoprenoid-derived lipid classes in prokaryotes that have been proposed to have similar membrane ordering properties as sterols.Methylobacterium extorquenscontains hopanoids and carotenoids in their outer membrane, making them an ideal system to investigate whether isoprenoid lipids play a complementary role in outer membrane ordering and cellular fitness. By genetically knocking outhpnE, andcrtBwe disrupted the production of squalene, and phytoene inMethylobacterium extorquensPA1, which are the presumed precursors for hopanoids and carotenoids, respectively. Deletion ofhpnEunexpectedly revealed that carotenoid biosynthesis utilizes squalene as a precursor resulting in a pigmentation with a C30backbone, rather than the previously predicted C40phytoene-derived pathway. We demonstrate that hopanoids but not carotenoids are essential for growth at high temperature. However, disruption of either carotenoid or hopanoid synthesis leads to opposing effects on outer membrane lipid packing. These observations show that hopanoids and carotenoids may serve complementary biophysical roles in the outer membrane. Phylogenetic analysis suggests thatM. extorquensmay have acquired the C30pathway through lateral gene transfer with Planctomycetes. This suggests that the C30carotenoid pathway may have provided an evolutionary advantage toM. extorquens.

Importance

All cells have a membrane that delineates the boundary between life and its environment. To function properly, membranes must maintain a delicate balance of physical and chemical properties. Lipids play a crucial role in tuning membrane properties. In eukaryotic organisms from yeast to mammals, sterols are essential for assembling a cell surface membrane that can support life. However, bacteria generally do not make sterols, so how do they solve this problem? Hopanoids and carotenoids are two major bacterial lipids, that are proposed as sterol surrogates. In this study we explore the bacteriumM. extorquensfor studying the role of hopanoids and carotenoids in surface membrane properties and cellular growth. Our findings suggest that hopanoids and carotenoids may serve complementary roles balancing outer membrane properties, and provide a foundation for elucidating the principles of surface membrane adaptation.
Aggregation and immobilisation of membrane proteins interplay with local lipid order in the plasma membrane of T cells
Authors: Urbančič Iztok , Schiffelers Lisa , Jenkins Edward , Gong Weijian , Santos Ana Mafalda , Schneider Falk , O’Brien-Ball Caitlin , Vuong Mai Tuyet , Ashman Nicole , Sezgin Erdinc , Eggeling Christian
Published: December 2020

The quest for understanding of numerous vital membrane-associated cellular processes, such as signalling, has largely focussed on the spatiotemporal orchestration and reorganisation of the identified key proteins, including their binding and aggregation. Despite strong indications of the involvement of membrane lipid heterogeneities, historically often termed lipid rafts, their roles in many processes remain controversial and mechanisms elusive. Taking activation of T lymphocytes as an example, we here investigate membrane properties around the key proteins – in particular the T cell receptor (TCR), its main kinase Lck, and phosphatase CD45. We determine their partitioning and co-localisation in passive cell-derived model membranes (i.e. giant plasma-membrane vesicles, GPMVs), and explore their mobility and local lipid order in live Jurkat T cells using fluorescence correlation spectroscopy and spectral imaging with polarity-sensitive membrane probes. We find that upon aggregation and partial immobilisation, the TCR changes its preference towards more ordered lipid environments, which can in turn passively recruit Lck. We observe similar aggregation-induced local membrane ordering and recruitment of Lck also by CD45, as well as by a membrane protein of antigen-presenting cells, CD86, which is not supposed to interact with Lck directly. This highlights the involvement of lipid-mediated interactions and suggests that the cellular membrane is poised to modulate the frequency of protein encounters according to their aggregation state and alterations of their mobility, e.g. upon ligand binding.
Development of functional corticomuscular connectivity from childhood to adulthood during precision grip with the dominant and non-dominant hand
Authors: Beck Mikkel Malling , Spedden Meaghan Elizabeth , Lundbye-Jensen Jesper
Published: December 2020

How does the neural control of manual movements mature from childhood to adulthood? Here, we investigated developmental differences in functional corticomuscular connectivity using coherence techniques in 91 individuals recruited from four different age groups covering the age range 8-30y. EEG and EMG were recorded while participants performed a unimanual visual force-tracing task requiring fine control of the force produced in a precision grip with both the dominant and non-dominant hand. Using beamforming methods, we reconstructed source activity from EEG data displaying peak coherence with the active FDI muscle during the task in order to assess functional corticomuscular connectivity. Our results revealed that coherence was greater in adolescents and adults than in children and that the difference in coherence between children and adults was driven by a greater magnitude of descending (cortex-to-muscle) coherence. This was paralleled by the observation of a posterior-to-anterior shift in the cortical sources displaying corticomuscular coherence within the contralateral hemisphere from late adolescence. Finally, we observed that corticomuscular coherence was higher on the non-dominant compared to the dominant hand across age groups. These findings provide a detailed characterization of differences in task-related corticomuscular connectivity for individuals at different stages of typical ontogenetic development that may be related to the maturational refinement of dexterous motor control.

Key points

‐

Fine motor control is gradually refined during human motor development, but little is known about the underlying neurophysiological mechanisms.

‐

Here, we used EEG and EMG to investigate functional corticomuscular connectivity during a precision grip with the dominant and non-dominant hand in 91 typically developed children, adolescents and adults (age range 8-30y).

‐

We show that older adolescents and adults are characterized by greater levels of corticomuscular coherence compared to children and that this is mainly driven by greater magnitudes of descending coherence (cortex-to-muscle). This is paralleled by a more anterior cortical site of coherence in older adolescents and adults compared to younger individuals.

‐

These results help us better understand the ontogenetic development of task-related functional connectivity in sensorimotor networks.
Human Polyomavirus-Encoded Circular RNAs
Authors: Yang Rong , Lee Eunice E. , Kim Jiwoong , Choi Joon H. , Chen Yating , Crewe Clair , Scherer Philipp E. , Kolitz Elysha , Cockerell Clay , Smith Taylor R. , Rosen Leslie , Verlinden Louisa , Feltkamp Mariet C. , Sullivan Christopher S. , Wang Richard C.
Published: December 2020

Circular RNAs (circRNAs) are a conserved class of RNAs with diverse functions. A subset of circRNAs are translated into peptides. Here we describe circular RNAs encoded by human polyomaviruses (HPyVs), including circular forms of RNAs encoding variants of the previously described alternative large T antigen open reading frame (ALTO) gene. Circular ALTO RNAs (circALTOs) can be detected in virus positive Merkel cell carcinoma (VP-MCC) cell lines and tumor samples. CircALTOs are stable, predominantly located in the cytoplasm, and N6-methyladenosine (m6A) modified. MCPyV circALTOs produce ALTO protein in cultured cells. MCPyV ALTO promotes the transcription of co-transfected reporter genes. MCPyV circALTOs are enriched in exosomes derived from VP-MCC lines and circALTO-transfected 293T cells, and purified exosomes can mediate ALTO expression and transcriptional activation. The related trichodysplasia spinulosa polyomavirus (TSPyV) also expresses a circALTO that can be detected in infected tissues and produces ALTO protein in cultured cells. Thus, human polyomavirus circRNAs are expressed in human tumors and tissues, encode for proteins, and may contribute to the infectious and tumorigenic properties of these viruses.
Transcriptional and epi-transcriptional dynamics of SARS-CoV-2 during cellular infection
Authors: Chang Jessie J.-Y. , Rawlinson Daniel , Pitt Miranda E. , Taiaroa George , Gleeson Josie , Zhou Chenxi , Mordant Francesca L. , Paoli-Iseppi Ricardo De , Caly Leon , Purcell Damian F.J. , Stinear Tim P. , Londrigan Sarah L. , Clark Michael B. , Williamson Deborah A. , Subbarao Kanta , Coin Lachlan J.M.
Published: December 2020

SARS-CoV-2 uses subgenomic (sg)RNA to produce viral proteins for replication and immune evasion. We applied long-read RNA and cDNA sequencing toin vitrohuman and primate infection models to study transcriptional dynamics. Transcription-regulating sequence (TRS)-dependent sgRNA was upregulated earlier in infection than TRS-independent sgRNA. An abundant class of TRS-independent sgRNA consisting of a portion of ORF1ab containingnsp1joined to ORF10 and 3’UTR was upregulated at 48 hours post infection in human cell lines. We identified double-junction sgRNA containing both TRS-dependent and independent junctions. We found multiple sites at which the SARS-CoV-2 genome is consistently more modified than sgRNA, and that sgRNA modifications are stable across transcript clusters, host cells and time since infection. Our work highlights the dynamic nature of the SARS-CoV-2 transcriptome during its replication cycle. Our results are available via an interactive web-app athttp://coinlab.mdhs.unimelb.edu.au/.
Interplay between extreme drift and selection intensities favors the fixation of beneficial mutations in selfing maize populations
Authors: Desbiez-Piat Arnaud , Rouzic Arnaud Le , Tenaillon Maud I. , Dillmann Christine
Published: December 2020

Population and quantitative genetic models provide useful approximations to predict long-term selection responses sustaining phenotypic shifts, and underlying multilocus adaptive dynamics. Valid across a broad range of parameters, their use for understanding the adaptive dynamics of small selfing populations undergoing strong selection intensity (thereafter High Drift-High selection regime, HDHS) remains to be explored. Saclay Divergent Selection Experiments (DSEs) on maize flowering time provide an interesting example of populations evolving under HDHS, with significant selection responses over 20 generations in two directions. We combined experimental data from Saclay DSEs, forward individual-based simulations, and theoretical predictions to dissect the evolutionary mechanisms at play in the observed selection responses. We asked two main questions: How do mutations arise, spread, and reach fixation in populations evolving under HDHS ? How does the interplay between drift and selection influence observed phenotypic shifts ? We showed that the long-lasting response to selection in small populations is due to the rapid fixation of mutations occurring during the generations of selection. Among fixed mutations, we also found a clear signal of enrichment for beneficial mutations revealing a limited cost of selection. Both environmental stochasticity and variation in selection coefficients likely contributed to exacerbate mutational effects, thereby facilitating selection grasp and fixation of small-effect mutations. Together our results highlight that despite a small number of polymorphic loci expected under HDHS, adaptive variation is continuously fueled by a vast mutational target. We discuss our results in the context of breeding and long-term survival of small selfing populations.
Ultra-high sensitivity mass spectrometry quantifies single-cell proteome changes upon perturbation
Authors: Brunner Andreas-David , Thielert Marvin , Vasilopoulou Catherine G. , Ammar Constantin , Coscia Fabian , Mund Andreas , Hoerning Ole B. , Bache Nicolai , Apalategui Amalia , Lubeck Markus , Richter Sabrina , Fischer David S. , Raether Oliver , Park Melvin A. , Meier Florian , Theis Fabian J. , Mann Matthias
Published: December 2020

Single-cell technologies are revolutionizing biology but are today mainly limited to imaging and deep sequencing1–3. However, proteins are the main drivers of cellular function and in-depth characterization of individual cells by mass spectrometry (MS)-based proteomics would thus be highly valuable and complementary4,5. Chemical labeling-based single-cell approaches introduce hundreds of cells into the MS, but direct analysis of single cells has not yet reached the necessary sensitivity, robustness and quantitative accuracy to answer biological questions6,7. Here, we develop a robust workflow combining miniaturized sample preparation, very low flow-rate chromatography and a novel trapped ion mobility mass spectrometer, resulting in a more than ten-fold improved sensitivity. We accurately and robustly quantify proteomes and their changes in single, FACS-isolated cells. Arresting cells at defined stages of the cell cycle by drug treatment retrieves expected key regulators such as CDK2NA, the E2 ubiquitin ligase UBE2S, DNA topoisomerases TOP2A/B and the chromatin regulator HMGA1. Furthermore, it highlights potential novel ones and allows cell phase prediction. Comparing the variability in more than 430 single-cell proteomes to transcriptome data revealed a stable core proteome despite perturbation, while the transcriptome appears volatile. This emphasizes substantial regulation of translation and sets the stage for its elucidation at the single cell level. Our technology can readily be applied to ultra-high sensitivity analyses of tissue material8, posttranslational modifications and small molecule studies to gain unprecedented insights into cellular heterogeneity in health and disease.
A neuronal circuit for vector computation builds an allocentric traveling-direction signal in theDrosophilafan-shaped body
Authors: Lyu Cheng , Abbott L.F. , Maimon Gaby
Published: December 2020

Many behavioral tasks require the manipulation of mathematical vectors, but, outside of computational models1–8, it is not known how brains perform vector operations. Here we show how theDrosophilacentral complex, a region implicated in goal-directed navigation8–14, performs vector arithmetic. First, we describe neural signals in the fan-shaped body that explicitly track a fly’sallocentrictraveling direction, that is, the traveling direction in reference to external cues. Past work has identified neurons inDrosophila12,15–17and mammals18,19that track allocentric heading (e.g., head-direction cells), but these new signals illuminate how the sense of space is properly updated when traveling and heading angles differ. We then characterize a neuronal circuit that rotates, scales, and adds four vectors related to the fly’segocentrictraveling direction–– the traveling angle referenced to the body axis––to compute the allocentric traveling direction. Each two-dimensional vector is explicitly represented by a sinusoidal activity pattern across a distinct neuronal population, with the sinusoid’s amplitude representing the vector’s length and its phase representing the vector’s angle. The principles of this circuit, which performs an egocentric-to-allocentric coordinate transformation, may generalize to other brains and to domains beyond navigation where vector operations or reference-frame transformations are required.
The anatomy of a phenological mismatch: interacting consumer demand and resource characteristics determine the consequences of mismatching
Authors: Wilde Luke R. , Simmons Josiah E. , Swift Rose J. , Senner Nathan R.
Published: December 2020

Climate change has caused shifts in seasonally recurring biological events and the temporal decoupling of consumer-resource pairs – i.e., phenological mismatching (hereafter, ‘mismatching’). Despite the hypothetical risk mismatching poses to consumers, it does not invariably lead to individual- or population-level effects. This may stem from how mismatches are typically defined, where an individual or population are ‘matched or mismatched’ based on the degree of asynchrony with a resource pulse. However, because both resource availability and consumer demands change over time, this categorical definition can obscure within- or among-individual fitness effects. We investigated the effects of resource characteristics on the growth, daily survival, and fledging rates of Hudsonian godwit (Limosa haemastica) chicks hatched near Beluga River, Alaska. To do this, we developed models to identify the effects of resource characteristics on individual- and population-level processes and determine how the strength of these effects change throughout a consumer’s early development. We found that at the individual-level, chick growth and survival improved following periods of higher invertebrate abundance but were increasingly dependent on the availability of larger prey as chicks aged. At the population level, seasonal fledging rates were best explained by a model including age-structured consumer demand. Our study suggests that modelling the effects of mismatching as a disrupted interaction between consumers and their resources provides a biological mechanism for how mismatching occurs and clarifies when it matters to individuals and populations. Given the variable responses to mismatching exhibited by consumer populations, such tools for predicting how populations may respond under future climatic conditions will be critical for conservation planning.
Characterizing intra-tumor and inter-tumor variability of immune cell infiltrates in murine syngeneic tumors
Authors: Mojtahedzadeh Sepideh , Opsahl Alan , Aguilar Joan-Kristel , Li Dingzhou , Streiner Nicole , Wang Jinwei , Trajkovic Dusko , Boucher Germaine , Coskran Timothy , O’Neil Shawn P. , Ram Sripad
Published: December 2020

By using serial-sectioning methodology combined with immunohistochemistry (IHC) and whole-slide digital image analysis (DIA), we present a systematic analysis of intra-tumor and inter-tumor variability in the abundance of nine immune cell biomarkers in multiple murine tumor models. Our analysis shows that inter-tumor variability is typically the dominant source of variation in measurements of immune cell densities. Statistical power analysis reveals how group size and variance in immune cell density estimates affect the predictive power for detecting a statistically meaningful fold-change in immune cell density. Inter-tumor variability in the ratio of immune cell densities show distinct patterns in select tumor models and reveal the existence of strong correlations between select biomarker pairs. Further, we show that the relative proportion of immune cells at different depths across tumor samples is preserved in some but not all tumor models thereby revealing the existence of compositional heterogeneity. The results and analyses presented here reveal the complex nature of immunologic heterogeneity that exists in murine tumor models and provide guidelines for designing preclinical studies for immuno-oncology research and drug development.
A WD40 repeat-like protein pathway connects F-BOX STRESS INDUCED (FBS) proteins to the NIGT1.1 transcriptional repressor in Arabidopsis
Authors: Sepulveda-Garcia Edgar , Fulton Elena C , Parlan Emily V , Brauning Ashley A , O’Connor Lily E , Fleming Anneke A , Replogle Amy J , Rocha-Sosa Mario , Gendron Joshua M , Thines Bryan
Published: December 2020

SCF-type E3 ubiquitin ligases use F-box (FBX) proteins as interchangeable substrate adaptors to recruit protein targets for ubiquitylation. FBX proteins almost universally have structure with two domains. A conserved N-terminal F-box domain interacts with a SKP protein and connects the FBX protein to the core SCF complex, while a C-terminal domain interacts with the protein target and facilitates recruitment. The F-BOX STRESS INDUCED (FBS) subfamily of four plant FBX proteins has atypical domain structure, however, with a centrally located F-box domain and additional conserved regions at both the N- and C-termini. FBS proteins have been linked to environmental stress networks, but no ubiquitylation target(s) or exact biological function has been established for this subfamily. We have identified two WD40 repeat-like proteins in Arabidopsis that are highly conserved in plants and interact with FBS proteins, which we have named FBS INTERACTING PROTEINs (FBIPs). FBIPs interact exclusively with the N-terminus of FBS proteins, and this interaction occurs in the nucleus. FBS1 destabilizes FBIP1, consistent with FBIPs being ubiquitylation targets of SCFFBScomplexes. Furthermore, we found that FBIP1 interacts with NIGT1.1, a GARP-type transcriptional repressor that regulates nitrate and phosphate starvation signaling and responses. Collectively, these interactions between FBS, FBIP, and NIGT1.1 proteins delineate a previously unrecognized SCF-connected transcription regulation module that works in the context of phosphate and nitrate starvation, and possibly other environmental stresses. Importantly, this work also identified two uncharacterized WD40 repeat-like proteins as new tools with which to probe how an atypical SCF complex, SCFFBS, functions via FBX protein N-terminal interaction events.
Creation and validation of 3D-printed head molds for stereotaxic injections of neonatal mouse brains
Authors: Chervonski Ethan , Brockman Asa A. , Khurana Rohit , Chen Yuhao , Greenberg Sabrina , Hay Madeleine S. , Luo Yifu , Miller Jason , Patelis Deanna , Whitney Sarah K. , Walker Matthew , Ihrie Rebecca A.
Published: December 2020

Background

An increasing number of rodent model systems use injection of DNA or viral constructs in the neonatal brain. However, approaches for reliable positioning and stereotaxic injection at this developmental stage are limited, typically relying on handheld positioning or molds that must be re-aligned for use in a given laboratory.

New method

A complete protocol and open source software pipeline for generating 3D-printed head molds derived from a CT scan of a neonatal mouse head cast, together with a universal adapter that can be placed on a standard stereotaxic stage.

Results

A series of test injections with adenovirus encoding red fluorescent protein were conducted using original clay molds and newly generated 3D printed molds. Several metrics were used to compare spread and localization of targeted injections.

Comparison with existing methods

The new method of head mold generation gave comparable results to the field standard, but also allowed the rapid generation of additional copies of each head mold with standardized positioning of the head each time.

Conclusions

This 3D printing pipeline can be used to rapidly develop a series of head molds with standardized injection coordinates across multiple laboratories. More broadly, this pipeline can easily be adapted to other perinatal ages or species.
Structural basis for the dimerization-dependent CRISPR-Cas12f nuclease
Authors: Xiao Renjian , Li Zhuang , Wang Shukun , Han Ruijie , Chang Leifu
Published: December 2020

Cas12f, also known as Cas14, is an exceptionally small type V-F CRISPR-Cas nuclease that is roughly half the size of comparable nucleases of this type. To reveal the mechanisms underlying substrate recognition and cleavage, we determined the cryo-EM structures of the Cas12f-sgRNA-target DNA and Cas12f-sgRNA complexes at 3.1 Å and 3.9 Å, respectively. An asymmetric Cas12f dimer is bound to one sgRNA for recognition and cleavage of dsDNA substrate with a T-rich PAM sequence. Despite its dimerization, Cas12f adopts a conserved activation mechanism among the type V nucleases which requires coordinated conformational changes induced by the formation of the crRNA-target DNA heteroduplex, including the close-to-open transition in the lid motif of the RuvC domain. Only one RuvC domain in the Cas12f dimer is activated by substrate recognition, and the substrate bound to the activated RuvC domain is captured in the structure. Structure-assisted truncated sgRNA, which is less than half the length of the original sgRNA, is still active for target DNA cleavage. Our results expand our understanding of the diverse type V CRISPR-Cas nucleases and facilitate potential genome editing applications using the miniature Cas12f.
Intra-Ramanome Correlation Analysis Unveils Metabolite Conversion Network from an Isogenic Cellular Population
Authors: He Yuehui , Huang Shi , Zhang Peng , Ji Yuetong , Xu Jian
Published: December 2020

Revealing dynamic features of cellular systems, such as links among metabolic phenotypes, typically requires a time- or condition-series set of samples. Here Intra-Ramanome Correlation analysis (IRCA) was proposed to achieve this goal from just one snapshot of an isogenic population, by pairwise correlating among cells all the thousands of Raman bands from Single-cell Raman Spectra (SCRS), i.e., based on the intrinsic inter-cellular metabolic heterogeneity. IRCA ofChlamydomonas reinhardtiiunder nitrogen depletion revealed a metabolite conversion network at each time point and its temporal dynamics that feature protein-to-starch conversion followed by starch-to-TAG conversion (plus conversion of membrane lipids to TAG). Such correlation patterns in IRCA were abrased by knocking out the starch-biosynthesis pathway yet fully restored by genetic complementation. Extension to 64 ramanomes from microalgae, fungi and bacteria under various conditions suggests IRCA-derived metabolite conversion network as an intrinsic, reliable, species-resolved and state-sensitive metabolic signature of isogenic cellular population. The high throughput, low cost, excellent scalability and broad extendibility of IRCA suggest its broad application in cellular systems.