bluuebunny/crossref_metadata_2025 huggingface dataset:
Specificity of SARS-CoV-2 antibody-detection assays against S and N protein among pre-COVID-19 sera from patients with protozoan and helminth parasitic infections
Authors: Yansouni Cedric P , Papenburg Jesse , Cheng Matthew P. , Corsini Rachel , Caya Chelsea , Camargo Fabio Vasquez , Harrison Luke B , Zaharatos Gerasimos , Büscher Philippe , Faye Babacar , Ndiaye Magatte , Matlashewski Greg , Ndao Momar
Published: August 2021

Background

We aimed to assess the specificity of SARS-CoV-2 antibody detection assays among people with known tissue-borne parasitic infections.

Methods

We tested three SARS-CoV-2 antibody-detection assays (cPass SARS-CoV-2 Neutralization Antibody Detection Kit, Abbott SARS-CoV-2 IgG assay, and STANDARD Q COVID-19 IgM/IgG Combo Rapid Test) among 559 pre-COVID-19 sera.

Results

The specificity of assays was 95-98% overall. However, lower specificity was observed among sera from patients with protozoan infections of the reticuloendothelial system, such as human African trypanosomiasis (Abbott Architect; 88% [95%CI 75-95]), visceral leishmaniasis (SD RDT IgG; 80% [95%CI 30-99]), and from patients with recent malaria from a holoendemic area of Senegal (ranging from 91% for Abbott Architect and SD RDT IgM to 98-99% for cPass and SD RDT IgG). For specimens from patients with evidence of past or present helminth infection overall, test specificity estimates were all ≥ 96%. Sera collected from patients clinically suspected of parasitic infections that tested negative for these infections yielded a specificity of 98-100%. The majority (>85%) of false-positive results were positive by only one assay.

Conclusions

The specificity of SARS-CoV-2 serological assays among sera from patients with tissue-borne parasitic infections was below the threshold required for decisions about individual patient care. Specificity is markedly increased by the use of confirmatory testing with a second assay. Finally, the SD RDT IgG proved similarly specific to laboratory-based assays and provides an option in low-resource settings when detection of anti-SARS-CoV-2 IgG is indicated.
Independently paced calcium oscillations in progenitor and differentiated cells in an ex vivo epithelial organ
Authors: Kim Anna A. , Nguyen Amanda , Marchetti Marco , Montell Denise J. , Pruitt Beth. L. , O’Brien Lucy Erin
Published: August 2021

Cytosolic calcium is a highly dynamic, tightly regulated, and broadly conserved cellular signal. Calcium dynamics have been studied widely in cellular monocultures, yet in vivo most organs comprise heterogeneous populations of stem and differentiated cells. We examined calcium dynamics in each cell type of the adult Drosophila intestine, a self-renewing epithelial organ where multipotent stem cells give rise to mature absorptive enterocytes and secretory enteroendocrine cells. Here we perform live imaging of whole organs ex vivo, and we employ orthogonal expression of red and green calcium sensors to determine whether calcium oscillations between different cell types are coupled. We show that stem cell daughters adopt strikingly distinct patterns of calcium oscillations when they acquire their terminal fates: Enteroendocrine cells exhibit single-cell calcium oscillations, while enterocytes exhibit rhythmic, long-range calcium waves. These multicellular waves do not propagate through progenitor cells (stem cells and enteroblasts), whose oscillation frequency is approximately half that of enteroendocrine cells. Organ-scale inhibition of gap junctions eliminates calcium oscillations in all three cell types, even, intriguingly, in progenitor and enteroendocrine cells that are surrounded only by enterocytes. Our findings establish that cells adopt fate-specific modes of calcium dynamics as they terminally differentiate and reveal that the oscillatory dynamics of different cell types in a single, coherent epithelium are paced independently.
The rate of ecosystem acclimation is the dominant uncertainty in long-term projections of an ecosystem service
Authors: Felton Andrew J. , Shriver Robert K. , Stemkovski Michael , Bradford John B. , Suding Katharine N. , Adler Peter B.
Published: August 2021

Rapid climate change may exceed ecosystems’ capacity to respond through processes including phenotypic plasticity, compositional turnover and evolutionary adaption. However, research predicting impacts of climate change on ecosystem services rarely consider this rate of “ecosystem acclimation.” Combining statistical models fit to historical climate data and remotely-sensed estimates of herbaceous productivity with an ensemble of climate models, we demonstrate that assumptions concerning acclimation rates are a dominant source of uncertainty: models assuming minimal acclimation project widespread decreases in forage production in the western US by 2100, while models assuming that acclimation keeps pace with climate change project widespread forage increases. Uncertainty related to ecosystem acclimation is larger than uncertainties from variation among climate models or emissions pathways. A better understanding of ecosystem acclimation is essential to improve long-term forecasts of ecosystem services, and shows that management to facilitate ecosystem acclimation may be necessary to maintain ecosystem services at historical baselines.
Sticky Pi, a high-frequency smart trap to study insect circadian activity under natural conditions
Authors: Geissmann Quentin , Abram Paul K. , Wu Di , Haney Cara H. , Carrillo Juli
Published: August 2021

In the face of severe environmental crises that threaten insect biodiversity, new technologies are imperative to monitor both the identity and ecology of insect species. Traditionally, insect surveys rely on manual collection of traps, which provide abundance data but mask the large intra- and inter-day variations in insect activity, an important facet of their ecology. Although laboratory studies have shown that circadian processes are central to insects’ biological functions, from feeding to reproduction, we lack the high-frequency monitoring tools to study insect circadian biology in the field. To address these issues, we developed the Sticky Pi, a novel, autonomous, open-source, insect trap that acquires images of sticky cards every twenty minutes. Using custom deep-learning algorithms, we automatically and accurately scored where, when and which insects were captured. First, we validated our device in controlled laboratory conditions with a classic chronobiological model organism, Drosophila melanogaster. Then, we deployed an array of Sticky Pis to the field to characterise the daily activity of an agricultural pest, Drosophila suzukii, and its parasitoid wasps. Finally, we demonstrate the wide scope of our smart trap by describing the sympatric arrangement of insect temporal niches in a community, without targeting particular taxa a priori. Together, the automatic identification and high sampling rate of our tool provide biologists with unique data that impacts research far beyond chronobiology; with applications to biodiversity monitoring and pest control as well as fundamental implications for phenology, behavioural ecology, and ecophysiology. We released the Sticky Pi project as an open community resource on https://doc.sticky-pi.com.
Identification of Subpallial Neuronal Populations Across Zebrafish Larval Stages that Express Molecular Markers for the Striatum
Authors: Aguda Vernie , Chasiotis Helen , Riadi Indira , Thiele Tod
Published: August 2021

Striatal neurons play a central role in vertebrate action selection; however, their location in larval zebrafish is not well defined. We assayed for conserved striatal markers in the zebrafish subpallium using fluorescentin situhybridization (FISH) and immunohistochemistry. Whole mount FISH revealed an inhibitory neuronal cluster rostral to the anterior commissure that expressestac1, a gene encoding substance P. This molecular profile is shared by mammalian striatal direct pathway neurons. A second partially overlapping population of inhibitory neurons was identified that expressespenka, a gene encoding enkephalin. This molecular profile is shared by striatal indirect pathway neurons. Immunostaining for substance P and enkephalin confirmed the presence of these peptides in the subpallium. Thetac1andpenkapopulations were both found to increase linearly across larval stages. Together, these findings support the existence of a striatal homologue in larval zebrafish that grows to match the development and increasing behavioural complexity of the organism.
The transmission of African Animal Trypanosomiasis (Nagana) in two districts in the forest zone of Ghana
Authors: Tweneboah Austine , Rosenau Jana , Agyapong Addo Kofi , Addison Thomas Kwame , Moussa Ibrahim Mahamat Alhadj , Weber Judith Sophie , Kelm Soerge , Badu Kingsley
Published: August 2021

The African Animal Trypanosomiasis (nagana) is caused by several species of trypanosome species which results in significant clinical diseases. This consequently leads to huge economic losses. We carried out a cross-sectional survey to investigate the composition of vectors, parasite diversity and intensities in two districts in the Eastern region of Ghana. Cytochrome c oxidase subunit 1 (COI) and internal transcribed spacer (ITS1) dependent PCR assays identified tsetse fly species and trypanosome parasites circulating in the area. A total of 229 tsetse flies, 65 pigs and 20 were sampled. Female and male flies 155 (51.8%), 74 (32.3%) respectively were collected with biconical traps. The overall vector density of 4.3 flies/trap/day was observed. Typanosome prevalence of 58.9% (95% CI: 52.5–65.1), 46.2% (95% CI: 34.6–58.1) and 0.0% (95% CI: 0.0–16.1) in tsetse flies, pigs and cattle respectively were detected.Trypanosoma congolensewas the predominant species with a prevalence of 80.7% (95% CI: 73.3–86.5) in flies and 60.0% (95% CI: 42.3–75.4). There was evidence of multiple trypanosome infection withT. congolense/T. simiaeoccurring highest with prevalence of 38.0% (95% CI: 30.7–46.9). The parasite prevalence in pigs across the communities was high with significant differences associated between locations (Chi2= 28.06, 95% CI: 0.05–0.81, P=0.0009). Blood meal with flies revealed feeding on both domesticSus scrofa domesticus(pig) andPhacochoerus africanus(warthog). Tsetse flies in this area remain highly infected with trypanosomes and continue to transmit the parasites to livestock and human populations in the communities.
Infinite storage capacity of memory-like mechanisms in the hippocampus: a cryptographic perspective on animal’s caching and retrieval behaviors
Authors: Mordechay Sharon , Forkosh Oren
Published: August 2021

The brain’s extraordinary abilities are often associated with its ability to learn and adapt. But memory and plasticity have their limitations, especially when faced with tasks such as retrieving thousands of food items such as in the case of scatter-hoarding animals. Here, we suggest a brain mechanism that works by utilizing cryptographic principles in lieu of plasticity. Rather than memorizing the locations of their caches, as previously suggested, we propose that cache-hoarding animals use a single cryptographic-like mechanism for both caching and retrieval. The mathematical model we developed functions similarly to hippocampal spatial cells, which respond to an animal’s positional attention. We know that the region that activates each spatial cell remains consistent across subsequent visits to the same area but not between areas. This remapping, combined with the uniqueness of cognitive maps, produces a persistent crypto-hash function for both food caching and retrieval. We show that our model is consistent with previous observations, such as animals’ ability to prioritize food items that are perishable or by their nutritional value. The model makes several measurable predictions regarding scattered hoarding and what factors can limit an animal’s retrieval success. Finally, while focusing here on scatter-hoarding, the mechanism we present might be utilized by the brain in other ways providing essentially infinite retention capacity for structured data.
The spatio-temporal program of liver zonal regeneration
Authors: Ben-Moshe Shani , Veg Tamar , Manco Rita , Dan Stav , Kolodziejczyk Aleksandra A. , Halpern Keren Bahar , Elinav Eran , Itzkovitz Shalev
Published: August 2021

The liver carries a remarkable ability to regenerate rapidly after acute zonal damage. Single-cell approaches are necessary to study this process, given the spatial heterogeneity of multiple liver cell types. Here, we use spatially-resolved single cell RNA sequencing (scRNAseq) to study the dynamics of mouse liver regeneration after acute acetaminophen (APAP) intoxication. We find that hepatocytes proliferate throughout the liver lobule, creating the mitotic pressure required to repopulate the necrotic pericentral zone rapidly. A subset of hepatocytes located at the regenerating front transiently up-regulate fetal-specific genes, including Afp and Cdh17, as they reprogram to a pericentral state. Zonated endothelial, hepatic-stellate cell (HSC) and macrophage populations are differentially involved in immune recruitment, proliferation and matrix remodeling. We observe massive transient infiltration of myeloid cells, yet stability of lymphoid cell abundance, in accordance with global decline in antigen presentation. Our study provides a resource for understanding the coordinated programs of zonal liver regeneration.
Evaluation of the speed, accuracy and precision of the QuickMIC rapid antibiotic susceptibility testing assay in a clinical setting
Authors: Malmberg Christer , Torpner Jessie , Fernberg Jenny , Öhrn Håkan , Johansson Cecilia , Tängdén Thomas , Kreuger Johan
Published: August 2021

The rapidly changing landscape of antimicrobial resistance poses a challenge for empirical antibiotic therapy in severely ill patients and highlights the need for fast antibiotic susceptibility diagnostics to guide treatment. Traditional methods for antibiotic susceptibility testing (AST) of bacteria such as broth microdilution (BMD) or the disc diffusion method (DDM) are comparatively slow and show high variability. Rapid AST methods under development often trade speed for resolution, sometimes only measuring responses at a single antibiotic concentration. QuickMIC is a recently developed lab-on-a-chip system for rapid AST. Here we evaluate the performance of the QuickMIC method with regard to speed, precision and accuracy in comparison to traditional diagnostic methods. 151 blood cultures of clinical Gram-negative isolates with a high frequency of drug resistance were tested with the QuickMIC system and compared with BMD for 12 antibiotics. To investigate sample turnaround time and functionality in a clinical setting, another 41 clinical blood culture samples were acquired from the Uppsala University Hospital and analyzed on site in the clinical laboratory with the QuickMIC system, and compared with DDM for 8 antibiotics routinely used in the clinical laboratory. The overall essential agreement between MIC values obtained by QuickMIC and BMD was 83.2%, with an average time to result of 3 h 2 min (SD: 24.8 min) for the QuickMIC method. For the clinical dataset, the categorical agreement was 94.9%, and the total turnaround time as compared to routine diagnostics reduced by 40% (33 h vs. 55 h). Interexperiment variability was low (average SD: 44.6% from target MIC) compared to the acceptable standard of ±1 log2unit (i.e. -50% to +100% deviation from target MIC) in BMD. We conclude that the QuickMIC method can provide rapid and accurate AST, and may be especially valuable in settings with high resistance rates and for antibiotics where wildtype and resistant MIC distributions are close or overlapping.
MODEL BASED OPTIMIZACION OF THE TERPENOIDS BIOSYNTHESIS INE. coli
Authors: Álvarez-Vasquez Fernando , González-Alcón Carlos , Gallego-Jara Julia , de Diego Teresa , Cánovas Manuel , Torres Néstor V.
Published: August 2021

Terpenoids are a family of compounds with high industrial interest and the development of biotechnological production methods is essential to achieve more sustainable alternatives to traditional extraction and synthesis methods. The modification and engineering of the catalytic activity (kcat) have been shown to be a feasible strategy in the biotechnological realm. Accordingly, we introduce a novel optimization strategy based in the modification of the kcat of the enzymes and applied it to the maximization of the terpenoids synthesis inE. coli. This approach is fairly general and can be applied alone or in conjunction with classic optimization strategies such as the modification of enzymatic specific activities.

For this purpose we first build up a reliable dynamic mathematical model of the alternative mevalonate pathway synthesis leading terpenoids biosynthesis inE. colithrough the methyl-D-erythritol 4-phosphate (MEP) pathway. This model includes the 2-C-methyl-D-erythritol 2, 4-diphosphate (MEC) pumps that mediate MEC extracellular extrusion. Although the physiological significance of the MEC extrusion is still discussed and their biological function is not clear, we find that this process is a must to guarantee bacteria homeostasis and cell viability.

We have identified the enzyme IspA as a bottleneck of the terpenoids biosynthesis, which is dual substrate for the two bisubstrate final reactions. Here are presented different ways to overcome this enzymatic flux restriction by modification of the IspA kcat or by introduction of new enzymes with parallel function.

Our results show that the MEP pathway optimized solutions for kcat can yield a maximum of 17.68 fold increment in the terpenoids biosynthetic flux when the kcat are modified. This maximal solution involves the modification of 8 kcat and the corresponding Km’s and Kd’s. Remarkably, this increase doesn’t imply a change in the total enzyme concentration of the cell. This is favorable output since enzyme overproduction can compromise cell functionality.

We also apply the overexpression of the enzyme activity approach and then we have compared and combined both strategies including scenarios as the deletion or import of the genes expressing enzymes not naturally present inE. coli.

A combined strategy of enzymes concentrations and kcat modifications with changes up ti six enzyme levels allows a flux increment of 21.22 fold the basal value. It involves the incorporation of two IspA with similar GPP and IPP bisubstrate functions than the native one but low affinity for the DMAPP as substrate.
DNAJB1-PRKACA in HEK293T cells induces LINC00473 overexpression that depends on PKA signaling
Authors: Kim Stephanie S. , Kycia Ina , Karski Michael , Ma Rosanna K. , Bordt Evan A. , Kwan Julian , Karki Anju , Winter Elle , Aktas Ranan G. , Wu Yuxuan , Emili Andrew , Bauer Daniel E. , Sethupathy Praveen , Vakili Khashayar
Published: August 2021

Fibrolamellar carcinoma (FLC) is a primary liver cancer that most commonly arises in adolescents and young adults in a background of normal liver tissue and has an poor prognosis due to lack of effective chemotherapeutic agents. The DNAJB1-PRKACA gene fusion (DP) has been reported in the majority of FLC tumors, however its oncogenic mechanisms remain unclear. Given the paucity of cellular models, in particular FLC tumor cell lines, we hypothesized that engineering the DP fusion gene in HEK293T cells would provide insight into the cellular effects of the fusion gene. We used CRISPR/Cas9 to engineer HEK293T clones expressing DP fusion gene (HEK-DP) and performed transcriptomic, proteomic, and mitochondrial studies to characterize this cellular model. Proteomic analysis of DP interacting partners identified mitochondrial proteins as well as proteins in other subcellular compartments. HEK-DP cells demonstrated significantly elevated mitochondrial fission, which suggests a role for DP in altering mitochondrial dynamics. Transcriptomic analysis of HEK-DP cells revealed a significant increase in LINC00473 expression, similar to what has been observed in primary FLC samples. LINC00473 overexpression was reversible with siRNA targeting of PRKACA as well as pharmacologic targeting of PKA and Hsp40 in HEK-DP cells. Therefore, our model suggests that LINC00473 is a candidate marker for DP activity.
RESTORING GLUTAMATE RECEPTOR SIGNALING IN PANCREATIC ALPHA CELLS RESCUES GLUCAGON RESPONSES IN TYPE 1 DIABETES
Authors: Panzer Julia K. , Tamayo Alejandro , Caicedo Alejandro
Published: August 2021

Glucagon secretion from pancreatic alpha cells is crucial to prevent hypoglycemia. For reasons still unknown, people with type 1 diabetes lose this glucoregulatory mechanism and are susceptible to dangerous hypoglycemia. Here we show that alpha cells in living pancreas slices from donors with type 1 diabetes failed to secrete glucagon in response to decreases in glucose concentration, thus mirroring the in vivo unresponsiveness to hypoglycemia. Glucagon content and responses to KCl depolarization were not affected, suggesting that alpha cells retained their secretory potential. By contrast, alpha cells had severely impaired signaling via glutamate receptors of the AMPA/kainate type. Under healthy conditions, activating these receptors was required to elicit full glucagon responses to decreases in glucose levels. In type 1 diabetes, reactivating residual glutamate receptor function with the positive allosteric modulators cyclothiazide and aniracetam restored glucagon secretion in response to hypoglycemia. These positive allosteric modulators are already approved to treat other conditions and could be repurposed to prevent hypoglycemia and improve management of diabetes.
Exon-Skipping Antisense Oligonucleotides for Cystic Fibrosis Therapy
Authors: Kim Young Jin , Sivetz Nicole , Layne Jessica , Voss Dillon , Yang Lucia , Zhang Qian , Krainer Adrian R.
Published: August 2021

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF), and theCFTR-W1282Xnonsense mutation causes a severe form of CF. Although Trikafta and other CFTR-modulation therapies benefit most CF patients, targeted therapy for patients with the W1282X mutation is lacking. The CFTR-W1282X protein has residual activity, but is expressed at a very low level due to nonsense-mediated mRNA decay (NMD). NMD-suppression therapy and read-through therapy are actively being researched forCFTRnonsense mutants. NMD suppression could increase the mutantCFTRmRNA, and read-through therapies may increase the levels of full-length CFTR protein. However, these approaches have limitations and potential side effects: because the NMD machinery also regulates the expression of many normal mRNAs, broad inhibition of the pathway is not desirable; and read-through drugs are inefficient, partly because the mutant mRNA template is subject to NMD. To bypass these issues, we pursued an exon-skipping antisense oligonucleotide (ASO) strategy to achieve gene-specific NMD evasion. A cocktail of two splice-site-targeting ASOs induced the expression ofCFTRmRNA without the PTC-containing exon 23 (CFTR-Δex23), which is an in-frame exon. Treatment of human bronchial epithelial cells with this cocktail of ASOs that target the splice sites flanking exon 23 results in efficient skipping of exon 23 and an increase in CFTR-Δex23 protein. The splice-switching ASO cocktail increases the CFTR-mediated chloride current in human bronchial epithelial cells. Our results set the stage for developing an allele-specific therapy for CF caused by the W1282X mutation.
A single shot of a hybrid hAdV5-based anti-COVID-19 vaccine induces a long-lasting immune response and broad coverage against VOC
Authors: López M. Verónica , Vinzón Sabrina E. , Cafferata Eduardo G. A. , Nuñez Felipe J. , Soto Ariadna , Sanchez-Lamas Maximiliano , Afonso Jimena , Aguilar-Cortes Diana , Ríos Gregorio D. , Maricato Juliana T. , Torres-Braconi Carla , Silveira Vanessa Barbosa-da , Montes-de Andrade Tatiane , Carvalho-de Souza Bonetti Tatiana , Ramos Janini Luiz M. , Castello Girão Manoel J. B. , Llera Andrea S. , Gomez Karina , Ortega Hugo H. , Berguer Paula M. , Podhajcer Osvaldo L.
Published: August 2021

Most approved vaccines against COVID-19 have to be administered in a prime/boost regimen. We engineered a novel vaccine based on a chimeric hAdV5 vector. The vaccine (named CoroVaxG.3) is based on three pillars: i) high expression of Spike to enhance its immunodominance by using a potent promoter and a mRNA stabilizer; ii) enhanced infection of muscle and dendritic cells by replacing the fiber knob domain of hAdV5 by hAdV3; iii) use of Spike stabilized in a prefusion conformation. Transduction with CoroVaxG.3 expressing Spike (D614G) dramatically enhanced Spike expression in human muscle cells, monocytes and dendritic cells compared to CoroVaxG.5 that expressed the native fiber knob domain. A single dose of CoroVaxG.3 induced potent humoral immunity with a balanced Th1/Th2 ratio and potent T-cell immunity, both lasting for at least 5 months. Sera from CoroVaxG.3 vaccinated mice was able to neutralize pseudoviruses expressing B.1 (wild type D614G), B.1.117 (alpha) and P.1 (gamma) Spikes, as well as an authentic WT and P.1 SARS-CoV-2 isolates. Neutralizing antibodies did not wane even after 5 months making this kind of vaccine a likely candidate to enter clinical trials
A CCaMK/Cyclops response element in the promoter ofL. japonicus Calcium-Binding Protein 1(CBP1) mediates transcriptional activation in root symbioses
Authors: Gong Xiaoyun , Jensen Elaine , Bucerius Simone , Parniske Martin
Published: August 2021

Early gene expression in arbuscular mycorrhiza (AM) and the nitrogen-fixing root nodule symbiosis (RNS) is governed by a shared regulatory complex. Yet many symbiosis-induced genes are specifically activated in only one of the two symbioses. TheLotus japonicusT-DNA insertion line T90, carrying a promoterlessuidA(GUS) gene in the promoter ofCalcium Binding Protein1(CBP1) is exceptional as it exhibits GUS activity in both root endosymbioses. To identify the responsiblecis-andtrans-acting factors, we subjected deletion/modification series ofCBP1promoter:reporter fusions to transactivation and spatio-temporal expression analysis and screened EMS-mutagenized T90 populations for aberrantGUSexpression. We identified onecis-regulatory element required forGUSexpression in the epidermis and a second element, necessary and sufficient for transactivation by the Calcium and Calmodulin-dependent protein kinase (CCaMK) in combination with the transcription factor Cyclops and conferring gene expression during both AM and RNS. Lack ofGUSexpression in T90whitemutants could be traced to DNA hypermethylation detected in and around this element. We concluded that the CCaMK/Cyclops complex can contribute to at least three distinct gene expression patterns on its direct target promotersNIN(RNS),RAM1(AM), andCBP1(AM and RNS), calling for yet-to-be identified specificity-conferring factors.
Human Deep Cortical Neurons Promote Regeneration and Recovery After Cervical Spinal Cord Injury
Authors: Doulames Vanessa M. , Weimann James , Plant Giles W.
Published: August 2021

Cervical spinal cord injuries (SCI) sever and permanently disrupt sensorimotor neural circuitry. Restoring connectivity within the damaged circuitry is critical to improving function. Herein we report robust regeneration of severed neural circuitry in a rat SCI model following transplantation of human induced pluripotent cells differentiated towards a deep cortical neuron lineage (iPSC-DCNs).In vivo, iPSC-DCNs: (1) integrated within the damaged cord and extended axons to caudal targets, (2) reversed SCI pathophysiology, (3) promoted robust regeneration of severed host supraspinal neural tracts, (4) and improved sensorimotor function. The results herein represent a significant paradigm shift in anatomical and functional outcomes over current preclinical/clinical models and demonstrates the survival and efficacy of human stem cell-derived cortical neurons in a SCI.
A gap-junction mutation in the mouse cochlea reveals cochlear amplification is driven by outer hair cell extracellular receptor potentials
Authors: Levic Snezana , Lukashkina Victoria A. , Simões Patricio , Lukashkin Andrei N. , Russell Ian J.
Published: August 2021

Cochlear amplification, whereby cochlear responses to low-to-moderate sound levels are amplified and compressed to loud sounds, is attributed to outer hair cell (OHC) electromotility driven by voltage changes across the OHC basolateral membranes due to sound-induced receptor-current modulation. Cochlear operation at high acoustic frequencies is enigmatic because the OHC intracellular receptor potential (RP) is severely attenuated at these frequencies. Clues to understanding the voltage control of OHC electromotility at different frequencies was provided by measurements from CD-1 mice with an A88V mutation of the gap-junction (GJ) protein connexin 30 (Cx30), which with Cx26, form heterogeneous GJs between supporting cells in the organ of Corti (OoC) and stria vascularis. The A88V mutation results in a smaller GJ conductance which may explain why the resistance across the OoC in CD-1Cx30A88V/A88Vmutants is higher compared with wild-type mice. The endocochlear potential, which drives the OHC receptor current and, consequently, the OHC RPs, is smaller in CD-1Cx30A88V/A88Vmutants. Even so, their high-frequency hearing sensitivity equals that of wild-type mice. Preservation of high-frequency hearing correlates with similar amplitude of extracellular receptor potentials (ERPs), measured immediately adjacent to the OHCs. ERPs are generated through OHC receptor current flow across the OoC electrical resistance, which is larger in CD-1Cx30A88V/A88Vthan in wild-type mice. Thus, smaller OHC receptor currents flowing across a larger OoC resistance in CD-1Cx30A88V/A88Vmice may explain why their ERP magnitudes are similar to wild-type mice. It is proposed that the ERPs, which are not subject to low-pass electrical filtering, drive high-frequency cochlear amplification.

Significance Statement

Cochlear amplification, whereby responses to low-to-moderate sound levels are amplified and those to loud sounds are compressed, is attributed to outer hair cell (OHC) electromotility. Electromotility is driven by voltage changes across the OHC basolateral membranes due to modulation of receptor current flow during sound-induced sensory hair bundle displacement. Mechanisms of high-frequency cochlear amplification remain to be elucidated. A mutation of the gap-junction protein connexin 30 decreases OHC intracellular receptor potentials in CD-1 mice. Instead of decreasing auditory sensitivity, the mutation rescues high-frequency hearing by causing OHC extracellular receptor potentials to be similar in amplitude to those of sensitive wild-type mice. It is proposed extracellular, not intracellular, potentials drive high-frequency OHC motility and cochlear amplification at high acoustic frequencies.
Circadian Clock Core Component Bmal1 Dictates Cell Cycle Rhythm of Proliferating Hepatocytes during Liver Regeneration
Authors: Jiang Huaizhou , Garcia Veronica , Yanum Jennifer Abla , Lee Joonyong , Dai Guoli
Published: August 2021

Following partial hepatectomy (PH), the majority of remnant hepatocytes synchronously enter and rhythmically progress through the cell cycle for three major rounds to regain lost liver mass. Whether and how the circadian clock core component Bmal1 modulates this process remains elusive. We performed PH onBmal1+/+and hepatocyte-specificBmal1knockout (Bmal1hep-/-) mice and compared the initiation and progression of the hepatocyte cell cycle. After PH, Bmal1+/+hepatocytes exhibited three major waves of nuclear DNA synthesis. In contrast, inBmal1hep-/-hepatocytes, the first wave of nuclear DNA synthesis was delayed by 12 h, and the third such wave was lost. Following PH, Bmal1+/+hepatocytes underwent three major waves of mitosis, whereasBmal1hep-/-hepatocytes fully abolished mitotic oscillation. These Bmal1-dependent disruptions in the rhythmicity of hepatocyte cell cycle after PH were accompanied by suppressed expression peaks of a group of cell cycle components and regulators, and dysregulated activation patterns of mitogenic signaling molecules c-Met and EGFR. Moreover,Bmal1+/+hepatocytes rhythmically accumulated fat as they expanded following PH, whereas this phenomenon was largely inhibited inBmal1hep-/-hepatocytes. In addition, during late stages of liver regrowth, Bmal1 absence in hepatocytes caused the activation of redox sensor Nrf2, suggesting an oxidative stress state in regenerated liver tissue. Collectively, we demonstrated that during liver regeneration, Bmal1 partially modulates the oscillation of S-phase progression, fully controls the rhythmicity of M-phase advancement, and largely governs fluctuations in fat metabolism in replicating hepatocytes, and eventually determines the redox state of regenerated livers.

New and Noteworthy

We demonstrated that Bmal1 centrally controls the synchronicity and rhythmicity of the cell cycle and lipid accumulation in replicating hepatocytes during liver regeneration. Bmal1 plays these roles, at least in part, by ensuring formation of the expression peaks of cell cycle components and regulators, as well as the timing and levels of activation of mitogenic signaling molecules.

Graphical Abstract
cDNA-detector: Detection and removal of cDNA contamination in DNA sequencing libraries
Authors: Qi Meifang , Nayar Utthara , Ludwig Leif S. , Wagle Nikhil , Rheinbay Esther
Published: August 2021

Exogenous cDNA introduced into an experimental system, either intentionally or accidentally, can appear as added read coverage over that gene in next-generation sequencing libraries derived from this system. If not properly recognized and managed, this cross-contamination with exogenous signal can lead to incorrect interpretation of research results. Yet, this problem is not routinely addressed in current sequence processing pipelines. Here, we present cDNA-detector, a computational tool to identify and remove exogenous cDNA contamination in DNA sequencing experiments. We apply cDNA-detector to several highly-cited public databases (TCGA, ENCODE, NCBI SRA) and show that contaminant genes appear in sequencing experiments where they lead to incorrect coverage peak calls. Our findings highlight the importance of sensitive detection and removal of contaminant cDNA from NGS libraries before downstream analysis.
RNA sequencing on muscle biopsy from a 5-week bedrest study reveals the effect of exercise and potential interactions with dorsal root ganglion neurons
Authors: McFarland Amelia J. , Ray Pradipta R. , Bhai Salman , Levine Benjamin , Price Theodore J.
Published: August 2021

Lack of physical activity is a predictor of poor health outcomes that can be prevented or reversed by exercise. Sedentary lifestyle, chronic disease or microgravity can cause muscle deconditioning that then has an impact on other physiological systems. An example is the nervous system, which is adversely affected by decreased physical activity resulting in increased incidence of neurological problems such as chronic pain. We sought to better understand how this might occur by conducting RNA sequencing experiments on muscle biopsies from human volunteers in a 5-week bed-rest study with an exercise intervention arm. We also used a computational method for examining ligand-receptor interactions between muscle and human dorsal root ganglion (DRG) neurons, the latter of which play a key role in nociception and are generators of signals responsible for chronic pain. We identified 1352 differentially expressed genes (DEGs) in bed rest subjects without an exercise intervention but only 132 DEGs in subjects with the intervention. Thirty-six genes were shared between the exercise and no intervention groups. Among 591 upregulated muscle genes in the no intervention arm, 26 of these were ligands that have receptors that are expressed by human DRG neurons. We detected a specific splice variant of one of these ligands, placental growth factor (PGF), in deconditioned muscle that binds to neuropilin 1, a receptor that is highly expressed in DRG neurons and known to promote neuropathic pain. We conclude that exercise intervention protects muscle from deconditioning transcriptomic changes, and prevents changes in expression of ligands that might sensitize DRG neurons that promote pain. Our work creates a set of actionable hypotheses to better understand how deconditioned muscle may influence the function of sensory neurons that innervate the entire body.
Structural basis of adenylyl cyclase 9 activation
Authors: Qi Chao , Lavriha Pia , Mehta Ved , Khanppnavar Basavraj , Mohammed Inayathulla , Li Yong , Lazaratos Michalis , Schaefer Jonas V. , Dreier Birgit , Plückthun Andreas , Bondar Ana-Nicoleta , Dessauer Carmen W. , Korkhov Volodymyr M.
Published: August 2021

Adenylyl cyclase 9 (AC9) is a membrane-bound enzyme that converts ATP into cAMP. The enzyme is weakly activated by forskolin, fully activated by the G protein Gαs subunit and is autoinhibited by the AC9 C-terminus. Although our recent structural studies of the AC9-Gαs complex provided the framework for understanding AC9 autoinhibition, the conformational changes that AC9 undergoes in response to activator binding remains poorly understood. Here, we present the cryo-EM structures of AC9 in several distinct states: (i) AC9 bound to a nucleotide inhibitor MANT-GTP, (ii) bound to an artificial activator (DARPin C4) and MANT-GTP, (iii) bound to DARPin C4 and a nucleotide analogue ATPαS, (iv) bound to Gαs and MANT-GTP. The artificial activator DARPin C4 partially activates AC9 by binding at a site that overlaps with the Gαs binding site. Together with the previously observed occluded and forskolin-bound conformations, structural comparisons of AC9 in the four new conformations show that secondary structure rearrangements in the region surrounding the forskolin binding site are essential for AC9 activation.

One Sentence Summary

Cryo-EM reveals activator-induced conformational changes in adenylyl cyclase AC9
Recording gene expression order in DNA by CRISPR addition of retron barcodes
Authors: Bhattarai-Kline Santi , Lear Sierra K. , Fishman Chloe B. , Lopez Santiago C. , Lockshin Elana R. , Schubert Max G. , Nivala Jeff , Church George , Shipman Seth L.
Published: August 2021

Biological processes depend on the differential expression of genes over time, but methods to make physical recordings of these processes are limited. Here we report a molecular system for making time-ordered recordings of transcriptional events into living genomes. We do this via engineered RNA barcodes, based on prokaryotic retrons1, which are reverse-transcribed into DNA and integrated into the genome using the CRISPR-Cas system2. The unidirectional integration of barcodes by CRISPR integrases enables reconstruction of transcriptional event timing based on a physical record via simple, logical rules rather than relying on pre-trained classifiers or post-hoc inferential methods. For disambiguation in the field, we will refer to this system as a Retro-Cascorder.
Experimental evidence that chronic outgroup conflict reduces reproductive success in a cooperatively breeding fish
Authors: Goncalves Ines Braga , Radford Andrew N.
Published: August 2021

Conflicts with conspecific outsiders are common in group-living species, from ants to primates, and are argued to be an important selective force in social evolution. However, whilst an extensive empirical literature exists on the behaviour exhibited during and immediately after interactions with rivals, only very few observational studies have considered the cumulative fitness consequences of outgroup conflict. Using a cooperatively breeding fish, the daffodil cichlid (Neolamprologus pulcher), we conducted the first experimental test of the effects of chronic outgroup conflict on reproductive investment and output. ‘Intruded’ groups received long-term simulated territorial intrusions by neighbours that generated consistent group-defence behaviour; matched ‘Control’ groups (each the same size and with the same neighbours as an Intruded group) received no intrusions in the same period. Intruded groups had longer inter-clutch intervals and produced eggs with less protein than Control groups. Despite this lower egg investment, Intruded groups provided more parental care, achieving similar hatching success to Control groups. Ultimately, however, Intruded groups had fewer and smaller surviving offspring than Control groups at 1-month post-hatching. We therefore provide experimental evidence that outgroup conflict can decrease fitness via cumulative effects on reproductive success, confirming the selective potential of this empirically neglected aspect of sociality.
The absence of the autoimmune regulator gene (AIRE) impairs the three-dimensional structure of medullary thymic epithelial cell spheroids
Authors: Monteleone-Cassiano Ana Carolina , Dernowsek Janaina A. , Mascarenhas Romario S. , Assis Amanda Freire , Pitol Dimitrius , Mardegan Issa João Paulo , Donadi Eduardo A. , Passos Geraldo Aleixo
Published: August 2021

Besides controlling the expression of peripheral tissue antigens, the autoimmune regulator (AIRE) gene also regulates the expression of adhesion genes in medullary thymic epithelial cells (mTECs), an essential process for mTEC-thymocyte interaction for triggering the negative selection in the thymus. For these processes to occur, it is necessary that the medulla compartment forms an adequate three-dimensional (3D) architecture, preserving the thymic medulla. Previous studies have shown thatAIREknockout (KO) mice have a small and disorganized thymic medulla; however, whetherAireinfluences the mTEC-mTEC interaction in the maintenance of the 3D structure has been little explored. Considering that AIRE controls cell adhesion genes, we hypothesized that this gene affects 3D mTEC-mTEC interaction. To test this, we constructed anin vitromodel system for mTEC spheroid formation, in which cells adhere to each other, establishing a 3D structure. The effect ofAireon mTEC-mTEC adhesion was evaluated by comparingAIREwild type (AIREWT)versus Aire KO (AIRE-/-)mTECs. Considering the 3D spheroid model evaluated, we reported that the absence ofAIREdisorganizes the 3D structure of mTEC spheroids, promotes a differential regulation of mTEC classical surface markers, and modulates genes encoding adhesion and other molecules.
Rampant transposition following RNAi loss causes hypermutation and antifungal drug resistance in clinical isolates of a human fungal pathogen
Authors: Priest Shelby J. , Yadav Vikas , Roth Cullen , Dahlmann Tim A. , Kück Ulrich , Magwene Paul M. , Heitman Joseph
Published: August 2021

Microorganisms survive and compete by stochastically acquiring mutations that enhance fitness. Although increased mutation rates are often deleterious in multicellular organisms, hypermutation can be beneficial for microbes experiencing strong selective pressures. Infections caused by Cryptococcus neoformans are responsible for ∼15% of AIDS-related deaths and associated with high mortality rates, attributable to a dearth of antifungal drugs and drug resistance. We identified two hypermutator C. neoformans clinical isolates in which Cnl1 transposon insertions were responsible for drug resistance. Whole-genome sequencing revealed both hypermutator genomes harbor a nonsense mutation in the RNAi component ZNF3 and hundreds of Cnl1 elements organized into massive subtelomeric arrays on every chromosome. QTL mapping identified a significant locus associated with hypermutation that included znf3. CRISPR-mediated editing of the znf3 nonsense mutation abolished hypermutation and restored siRNA production. In sum, hypermutation and drug resistance in these isolates results from RNAi loss and a significant burden of Cnl1 elements.
Single-cell lineage trajectories and chromatin regulators that initialize antiviral CD8 T cell ontogeny
Authors: Diao Huitian , Chen Runqiang , Tsuda Shanel M. , Wang Dapeng , Frederick Megan A. , Kim Jihye , Karunadharma Pabalu P. , Martinez Gustavo , Getzler Adam J. , Toma Clara , Milner Justin J. , Venables Thomas C. , Martin Donna M. , Goldrath Ananda W. , Crotty Shane , Pipkin Matthew E.
Published: August 2021

Individual naive CD8 T cells activated in lymphoid organs differentiate into functionally diverse and anatomically distributed T cell phylogenies in response to intracellular microbes. During infections that resolve rapidly, including live viral vaccines1, distinct effector (TEFF) and memory (TMEM) cell populations develop that ensure long term immunity2. During chronic infections, responding cells progressively become dysfunctional and “exhaust”3. A diverse taxonomy of TEFF, TMEM and exhausted (TEX) CD8 T cell populations is known, but the initial developmental basis of this phenotypic variation remains unclear4–10. Here, we defined single-cell trajectories and identified chromatin regulators that establish antiviral CD8 T cell heterogeneity using unsupervised analyses of single-cell RNA dynamics11–13 and an in vivo RNAi screen14. Activated naive cells differentiate linearly into uncommitted effector-memory progenitor (EMP) cells, which initially branch into an analogous manifold during either acute or chronic infection. Disparate RNA velocities in single EMP cells initiate divergence into stem, circulating, and tissue-resident memory lineages that generate diverse TMEM and TEX precursor states in specific developmental orders. Interleukin-2 receptor (IL-2R) signals are essential for formation and transcriptional heterogeneity of EMP cells, and promote trajectories toward TEFF rather than TEX states. Nucleosome remodelers Smarca4 and Chd7 differentially promote transcription that delineates divergent TMEM lineages before cooperatively driving terminal TEFF cell differentiation. Thus, the lineage architecture is established by specific chromatin regulators that stabilize diverging transcription in uncommitted progenitors.
Maternal and paternal sugar consumption interact to modify offspring life history and physiology
Authors: Camilleri-Carter Tara-Lyn , Piper Matthew D.W. , Robker Rebecca L. , Dowling Damian K.
Published: August 2021

Intergenerational effects on offspring phenotypes occur in response to variation in both maternal and paternal nutrition. Because the combined maternal and paternal effects are rarely considered together however, their relative contributions, and the capacity for interactions between parental diets to shape offspring life history and physiology are not understood. To address this, we altered sucrose levels of adult fruit flies (Drosophila melanogaster) prior to mating, across two generations, producing parent-parent and parent-offspring combinations that were either matched or mismatched in dietary sucrose. We then measured lifespan, fecundity, body mass, and triglyceride levels in parents and offspring. We reveal complex non-additive interactions, that involve diets of each parent and offspring to shape offspring phenotypes, but the effects were generally not consistent with an adaptive response to parental diet. Notably, we find that interacting parental flies (sires and dams) lived longer when their sucrose treatments were matched, but they produced shorter-lived offspring.