bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of different methods for in vitro susceptibility testing of colistin in carbapenem resistant Gram negative bacilli
Authors: Rout Bidyutprava , Dash Sumesh Kumar , Sahu Kundan kumar , Behera Birasen , Praharaj Ira , Otta Sarita
Published: August 2023

Introduction- The increasing antibiotic resistance like advent of carbapenem resistant
Enterobactarales (CRE), Carbapenem Resistant Acinetobacter baumanii (CRAB) and
Carbapenem Resistant Pseudomonas aeruginosa (CRPA) has led to use of toxic and older drugs
like colistin for these organisms. But worldwide there is an increase in resistance even to colistin
mediated both by chromosomes and plasmids. This necessitates accurate detection of resistance.
This is impeded by unavailability of a user friendly phenotypic method for use in routine clinical
microbiology practice. The present study attempts to evaluate two different methods- Colistin
broth disc elution and MIC detection by Vitek 2 in comparison to CLSI approved broth
microdilution (BMD) for colistin for Enterobactarales, Pseudomonas aeruginosa and
Acinetobacter baumanii clinical isolates.
Methods- Colistin susceptibility of 6013 carbapenem resistant isolates was determined by BMD,
Colistin Broth Disc Elution (CBDE) and Vitek2 methods and was interpreted as per CLSI
guidelines. The MIC results of CBDE, Vitek 2 were compared with that of BMD and essential
agreement (EA), categorical agreement (CA), sensitivity, specificity, very major error (VME),
major error (ME) and cohen’s kappa (CK) was calculated. Presence of any plasmid-mediated
colistin resistance (mcr-1, 2, 3, 4 and 5) was evaluated all colistin resistant isolates by
conventional polymerase chain reaction.
Results- Colistin resistance was found in 778 (12.9%) strains among the carbapenem resistant
isolates. Klebsiella pneumoniae had the highest (18.9%) colistin resistance by BMD method.
MIC of Vitek 2 had sensitivity ranging from 78.2% to 84.8% and specificity of > 92%. There
were 171 VMEs and 323 MEs by Vitek2 method, much more than CLSI acceptable range. The
highest percentage of errors was committed for Acinetobacter baumanii (27.8% of VME and
7.9% ME). On the other hand the CBDE method performed well with EA, CA, VME and ME
within acceptable range for all the organisms. The sensitivity of CBDE method compared to gold
standard BMD varied from 97.5%-98.8% for different strains with a specificity of more than
97.6%. None of the isolated colistin resistant organism harbored mcr plasmids.
Conclusion- As BMD has many technical complexities, CBDE is the best viable alternative
available for countries like India. A sensitive MIC reported by Vitek2 needs to be carefully
considered due high propensity for VMEs particularly for Klebsiella spp.
Data summary- Supplementary material provides data regarding the comparison of MICs of three different mmethods for all samples. Sheet 1 to Sheet 5 contains data for E.coli, K.pneumoniae,A.baumannii, P.aeruginosa, E. cloacae respectively.
Kaposi’s sarcoma herpesvirus viral FLICE inhibitory protein modulates A20 deubiquitinase activity
Authors: Herold Kevin , Ruffin Ayana , Chmura Jennifer C. , Dellomo Anna J. , Ehrlich ELANA
Published: August 2023

KSHV viral FLICE inhibitory protein (vFLIP) is a potent activator of NF-κB signaling and an inhibitor of apoptosis and autophagy. Inhibition of vFLIP function and NF-κB signaling promotes lytic reactivation. Here we provide evidence for a novel function of vFLIP through inhibition of the deubiquitinating (DUB) activity of the negative regulator, A20. We demonstrate direct interaction of vFLIP with the Itch/A20 ubiquitin editing complex and provide evidence for subsequent loss of A20 DUB activity. Our results provide further insight into the function of vFLIP in the regulation of NF-κB signaling.
Thiovibrio frasassiensis gen. nov., sp. nov., an autotrophic, elemental sulphur disproportionating bacterium isolated from sulphidic karst sediment, and proposal of Thiovibrionaceae fam. nov.
Authors: Aronson Heidi S. , Thomas Cais , Bhattacharyya Maia K. , Eckstein Shaan R. , Jensen Sophia R. , Barco Roman A. , Macalady Jennifer L. , Amend Jan P.
Published: August 2023

A novel, autotrophic, mesophilic bacterium, strain RS19-109T, was isolated from sulphidic stream sediments in the Frasassi Caves, Italy. The cells of this strain grew chemolithoautotrophically under anaerobic conditions while disproportionating elemental sulphur (S0) and thiosulphate, but not sulphite with bicarbonate/CO2 as a carbon source. Autotrophic growth was also observed with molecular hydrogen as an electron donor, and S0, sulphate, thiosulphate, nitrate and ferric iron as electron acceptors. Oxygen was not used as an electron acceptor and sulphide was not used as an electron donor. Weak growth was observed with sulphate as an electron acceptor and organic carbon as an electron donor and carbon source. The strain also showed weak growth by fermentation of tryptone. It grew at pH 5.5–7.5 (optimum, pH 7.0), 4–35 °C (optimum, 30 °C) and between 0–1.7 % NaCl. Strain RS19-109T was found to be phylogenetically distinct based on 16S rRNA gene sequence similarity (89.2 %) to its closest relative,

Desulfurivibrio alkaliphilus

AHT2T. The draft genome sequence for strain RS19-109T had average nucleotide identity, average amino acid identity and in silico DNA–DNA hybridization values of 72.2, 63.0 and 18.3 %, respectively, compared with the genome sequence of

D. alkaliphilus

AHT2T. On the basis of its physiological and genomic properties, strain RS19-109T is proposed as the type strain of a novel species of a novel genus, Thiovibrio frasassiensis gen. nov., sp. nov. A novel family, Thiovibrionaceae fam. nov., is proposed to accommodate Thiovibrio within the order

Desulfobulbales

. Strain RS19-109T has been deposited at the DSMZ-German Collection of Microorganisms and Cell Cultures (=DSM 115074T) and the American Type Culture Collection (=ATCC TSD-325T).
Acinetobacter entericus sp. nov., isolated from the gut of plastic-eating insect larvae Zophobas atratus
Authors: Dong Xuena , Yang Yu
Published: August 2023

A Gram-negative, non-motile and rod-shaped strain, BIT-DXN8T, was isolated from the gut of plastic-eating insect larvae Zophobas atratus. The taxonomic position of this new isolate was examined by using a polyphasic approach. A preliminary analysis based on the 16S rRNA gene sequence (1411 bp) indicated that the most similar strain to BIT-DXN8T was

Acinetobacter bouvetii

DSM 14964T (98.5%), followed by

Acinetobacter haemolyticus

CIP 64.3T (98.2%) and Acinetobacter pullicarnis S23T (98.2%). The results of phylogenetic analyses, based on the 16S rRNA gene, concatenated sequences of five housekeeping genes (fusA, gyrB, recA, rplB and rpoB) and genome sequences, placed strain BIT-DXN8T in a separate lineage among the genus

Acinetobacter

of the family

Moraxellaceae

. The average nucleotide identity and digital DNA–DNA hybridization values of the strain when compared to all other species within the genus

Acinetobacter

were below 96 and 70 %, respectively. The physiological and biochemical tests confirm the affiliation of strain BIT-DXN8T to the present species within the genus

Acinetobacter

, but with some specific phenotypic differences. Therefore, strain BIT-DXN8T is considered to represent a novel species, for which the name Acinetobacter entericus sp. nov. is proposed. The type strain is BIT-DXN8T (=CCTCC AB 2022117T=KCTC 92696T).
Survival trends among people living with human immunodeficiency virus on antiretroviral treatment in two rural districts in Ghana
Authors: Sackeya Eugene , Beru Martin Muonibe , Angmortey Richard Nomo , Boakye Kingsley , Baatira Musah , Yakubu Mohammed Sheriff , Opoku Douglas Aninng , Mohammed Aliyu , Ayisi-Boateng Nana Kwame , Boateng Daniel , Nakua Emmanuel Kweku , Edusei Anthony Kweku
Published: August 2023

Background

The human immunodeficiency virus (HIV) has caused a lot of havoc since the early 1970s, affecting 37.6 million people worldwide. The 90-90-90 treatment policy was adopted in Ghana in 2015 with the overall aim to end new infections by 2030, and to improve the life expectancy of HIV seropositive individuals. With the scale-up of Highly Active Antiretroviral Therapy (HAART), the lifespan of People Living with HIV (PLWH) on antiretrovirals (ARVs) is expected to improve. In rural districts in Ghana, little is known about the survival probabilities of PLWH on ARVs hence, this study was conducted to estimate the survival trends of PLWH on ARVs for the periods between 2016 to 2020.

Methods

A retrospective evaluation of data gathered across ARV centers within Tatale and Zabzugu districts in Ghana from 2016 to 2020 among PLWH on ARVs. The census technique was employed and a total of 261 participants were recruited for the study. The data was analyzed using STATA software version 16.0. Lifetable analysis and Kaplan-Meier graph were used to assess the survival probabilities. “Stptime” per 1000 person-years and the competing risk regression was used to evaluate mortality rates and risk of mortality.

Results

The cumulative survival probability was 0.8847 (95% CI: 0.8334-0.9209). The overall mortality rate was 51.89 (95% CI: 36.89-72.97) per 1000 person-years. WHO stage III and IV [AHR: 4.25 (95%CI: 1.6-9.71) p = 0.001] as well as age group (50+years) [AHR: 5.02 (95% CI: 1.78-14.13) p=0.002] were associated with mortality.

Conclusion

Survival probabilities is high among the population of PLWH in Ghana with declining mortality rates. Clinicians should provide critical attention and care to patients at HIV WHO stages III and IV and intensify HIV screening at all entry points since early diagnosis is associated with high survival probabilities.
Divergence of variant binding/neutralizing antibodies following SARS-CoV-2 booster vaccines in myeloma: Impact of hybrid immunity
Authors: Moreno Alberto , Manning Kelly , Azeem Maryam I. , Nooka Ajay K. , Ellis Madison , Manalo Renee Julia , Switchenko Jeffrey M. , Wali Bushra , Kaufman Jonathan L. , Hofmeister Craig C. , Joseph Nisha S. , Lonial Sagar , Dhodapkar Kavita M. , Dhodapkar Madhav V. , Suthar Mehul S.
Published: August 2023

We characterized virus-neutralization and spike-binding antibody profiles in myeloma patients following monovalent or bivalent-SARS-CoV-2 booster vaccination. Vaccination improves the breadth of binding antibodies but not neutralization activity against current variants. Hybrid immunity and immune imprinting impact vaccine-elicited immunity.
Latitudinal patterns of flowering phenology of three widespread tropical species from public media and data repositories
Authors: Kriti Bhavya , Sankaran Sidharth , Ramaswami Geetha
Published: August 2023

Plant phenology encompasses the study of periodic events in biological life cycles of plants and the influence of seasonal changes, climate, temperature and other environmental factors on these events. In order to understand the role of the environment and the changing climate on the life cycle of plants, data suggesting long term trends are required as a baseline to compare against future changes. This study aims to analyze the spatial flowering pattern of the plant species -Bombax ceiba, Butea monosperma and Cassia fistula across India, through images uploaded on public media repositories like citizen science portals and social media. Citizen science is rapidly emerging as a reliable source of supplementary scientific information, and can be utilized to understand broad patterns of tree behavior. We found that for all three species, flowering phenology is longer, and begins earlier in southern latitudes as compared to the northern latitudes. We conclude that publicly available images of flowering trees are a good source to supplement baseline data on tree phenology across India.
Multiplecis-regulatory elements controlprox1aexpression in distinct lymphatic vascular beds
Authors: Panara Virginia , Yu Hujun , Peng Di , Staxäng Karin , Hodik Monika , Filipek-Gorniok Beata , Kazenwadel Jan , Skoczylas Renae , Mason Elizabeth , Allalou Amin , Harvey Natasha L. , Haitina Tatjana , Hogan Benjamin M. , Koltowska Katarzyna
Published: August 2023

Lymphatic vessels play a role in several physiological and pathological processes including tissue fluid homeostasis, dietary fat absorption, immunosurveillance, and immunomodulation. During embryonic development, lymphatic endothelial cell (LEC) precursors are distinguished from blood endothelial cells by the expression of the transcription factor Prospero-related homeobox 1(PROX1). PROX1 is essential for lymphatic vascular network formation in mouse and zebrafish. The initiation of PROX1 expression precedes LEC sprouting and migration, serving as the definitive marker of specified LECs. Despite its crucial role in lymphatic development, the upstream regulation ofPROX1in LECs remains to be uncovered. SOX18 and COUP-TFII are thought to regulateProx1expression in mice by binding to its promoter region. However, how the specificity ofProx1expression to LECs is achieved remains to be studied in detail.

In this study, we analysed evolutionary conservation and chromatin accessibility to identify enhancer sequences located in the proximity of zebrafishprox1aactive in developing LECs. We confirmed the functional role of the identified sequences through CRISPR/Cas9 mutagenesis of a lymphatic valve enhancer. The deletion of this genomic region results in impaired valve morphology and function. Overall, our results reveal the intricate control ofprox1aexpression through a collection of enhancers. Ray-finned fish-specific distal enhancers drive pan-lymphatic expression, while vertebrate-conserved proximal enhancers refine expression in functionally distinct subsets of lymphatic vessels.

Graphical Abstract
Correlating stress reduction and eye movement patterns in a world famous Kyoto Japanese garden
Authors: Goto Seiko , Takase Hiroki , Yamaguchi Keita , Kato Tomoki , Sun Minkai , Koga Aoi , Tiankai Liang , Poy Isamu A. , Herrup Karl
Published: August 2023

Visual stimuli have been repeatedly shown to elicit significant non-visual responses. In a continuing effort to explore the unique effects of viewing a Japanese garden on the physiological and psychological metrics of stress, we gained access to the world famous Murin-an garden in Kyoto, Japan. This well-maintained observation garden was designed to be viewed while seated at a single vantage point to maximize the impact of the visual scene. As a control, we used a public garden on the campus of Kyoto University that was designed in a similar style. Sixteen college age students were asked to view both gardens while we monitored their pulse rates and tracked their eye movements. We used the POMS questionnaire to determine the effect of the garden viewing on the mood of the participants. We found that the Murin-an garden was more effective in decreasing pulse rate and improving mood than the University garden. The eye tracking data showed that during their Murin-an viewing the participants gaze ranged far more broadly across the visual field both the X-Y plane and in depth, and the speed with which the eyes moved from point-to-point was greater. Taken together, our data suggest that no one element in the garden was dominant in eliciting the changes in heart rate and mood. Rather, it was the breadth and rapidity of the shifts in gaze that drove the effects, a conclusion with implications for other interventions aimed stress reduction.

Significance

Views of nature and natural phenomena have a well-recognized calming effect on humans that has recognized therapeutic value in both medical and psychological settings. Our work explores the source of this effect by having participants view Murin-an, a world-famous Japanese style garden. Using both psychological and physiological measures, we confirm and extend earlier findings showing that a well constructed garden can effectively lower heart rate and improve mood within minutes. We also find, by analogy with eye movement desensitization and reprograming (EMDR), that it is participants’ rapidly shifting gaze rather than a single specific visual object is the most likely source of the calming effect.
Shh from mossy cells contributes to preventing NSC pool depletion after seizure-induced neurogenesis and in aging
Authors: Noguchi Hirofumi , Arela Jessica Chelsea , Ngo Thomas T. , Cocas Laura , Pleasure Samuel J.
Published: August 2023

Epileptic seizures induce aberrant neurogenesis from resident neural stem cells (NSCs) in the dentate gyrus of the adult mouse hippocampus, which has been implicated in depletion of the NSC pool and impairment of hippocampal function. However, the mechanisms regulating neurogenesis after seizures remain unknown. Here we demonstrate that Shh from mossy cells is a major source of Shh signaling activity after seizures, by which mossy cells contribute to seizure-induced neurogenesis and maintenance of the NSC pool. Deletion of Shh from mossy cells attenuates seizure-induced neurogenesis. Moreover, in the absence of Shh from mossy cells, NSCs pool are prematurely depleted after seizure-induced proliferation, and NSCs have impaired self-renewal. Likewise, lack of Shh from mossy cells accelerates age-related decline of the NSC pool with accompanying reduction of self-renewal of NSCs outside the context of pathology such as seizures. Together, our findings indicate that Shh from mossy cells is critical to maintain NSCs and to prevent exhaustion from excessive consumption in aging and after seizures.
Rule-based modulation of a sensorimotor transformation across cortical areas
Authors: Chang Yi-Ting , Finkel Eric A. , Xu Duo , O’Connor Daniel H.
Published: August 2023

Flexible responses to sensory stimuli based on changing rules are critical for adapting to a dynamic environment. However, it remains unclear how the brain encodes rule information and uses this information to guide behavioral responses to sensory stimuli. Here, we made single-unit recordings while head-fixed mice performed a cross-modal sensory selection task in which they switched between two rules in different blocks of trials: licking in response to tactile stimuli applied to a whisker while rejecting visual stimuli, or licking to visual stimuli while rejecting the tactile stimuli. Along a cortical sensorimotor processing stream including the primary (S1) and secondary (S2) somatosensory areas, and the medial (MM) and anterolateral (ALM) motor areas, the single-trial activity of individual neurons distinguished between the two rules both prior to and in response to the tactile stimulus. Variable rule-dependent responses to identical stimuli could in principle occur via appropriate configuration of pre-stimulus preparatory states of a neural population, which would shape the subsequent response. We hypothesized that neural populations in S1, S2, MM and ALM would show preparatory activity states that were set in a rule-dependent manner to cause processing of sensory information according to the current rule. This hypothesis was supported for the motor cortical areas by findings that (1) the current task rule could be decoded from pre-stimulus population activity in ALM and MM; (2) neural subspaces containing the population activity differed between the two rules; and (3) optogenetic disruption of pre-stimulus states within ALM and MM impaired task performance. Our findings indicate that flexible selection of an appropriate action in response to a sensory input can occur via configuration of preparatory states in the motor cortex.

HIGHLIGHTS

Task rules are reflected in preparatory activity in sensory and motor cortices.

Neural subspaces for processing tactile signals depend on the current task rule.

Motor cortical activity tracks rule switches and is required for flexible rule-guided behavior.
Efficient computation by molecular competition networks
Authors: Cai Haoxiao , Wei Lei , Zhang Xiaoran , Qiao Rong , Wang Xiaowo
Published: August 2023

Most biomolecular systems exhibit computation abilities, which are often achieved through complex networks such as signal transduction networks. Particularly, molecular competition in these networks can introduce crosstalk and serve as a hidden layer for cellular information processing. Despite the increasing evidence of competition contributing to efficient cellular computation, how this occurs and the extent of computational capacity it confers remain elusive. In this study, we introduced a mathematical model for Molecular Competition Networks (MCNs) and employed a machine learning-based optimization method to explore their computational capacity. Our findings revealed that MCNs, when compared to their non-competitive counterparts, demonstrate superior performance in both discrete decision-making and analog computation tasks. Furthermore, we examined how real biological constraints influence the computational capacity of MCNs, and highlighted the nonnegligible role of weak interactions. The study suggested the potential of MCNs as efficient computational structures in bothin vivoandin silicoscenarios, providing new insights into the understanding and application of cellular information processing.
Controlling biases in targeted plant removal experiments
Authors: Monteux Sylvain , Blume-Werry Gesche , Gavazov Konstantin , Kirchhoff Leah K. , Krab Eveline J. , Lett Signe , Pedersen Emily Pickering , Väisänen Maria
Published: August 2023

Targeted removal experiments are a powerful tool to assess the effects of plant species or (functional) groups on ecosystem functions. However, removing plant biomass in itself can bias the observed responses. This bias is commonly addressed by waiting until ecosystem recovery, but this is inherently based on unverified proxies or anecdotal evidence. Statistical control methods are efficient, but restricted in scope by underlying assumptions.

We propose accounting for such biases within the experimental design, using a gradient of biomass removal controls. We demonstrate the relevance of this design by presenting i) conceptual examples of suspected biases and ii) how to observe and control for these biases.

Using data from a mycorrhizal association-based removal experiment we show that ignoring biomass removal biases (including by assuming ecosystem recovery) can lead to incorrect, or even contrary conclusions (e.g., false positive and false negative). Our gradient design can prevent such incorrect interpretations, whether aboveground biomass has fully recovered or not.

Our approach provides more objective and quantitative insights, independently assessed for each variable, than using a proxy to assume ecosystem recovery. Our approach circumvents the strict statistical assumptions of e.g. ANCOVA and thus offers greater flexibility in data analysis.
Conserved switch genes regulate a novel cannibalistic morph after whole genome duplication
Authors: Wighard Sara , Witte Hanh , Sommer Ralf J.
Published: August 2023

Developmental plasticity facilitates morphological and behavioural novelty, but associated regulatory mechanisms remain elusive. Nematodes have emerged as a powerful model to study developmental plasticity and its evolution. Here, we show the predatory nematodeAllodiplogaster sudhausievolved an additional third mouth morph, concomitant with whole genome duplication (WGD) and a strong increase in body size. The three mouth morphs are induced by different diets; bacteria, fungi and nematodes. CRISPR experiments indicate that regulation of the third morph involves co-option of a conserved developmental switch gene, which through WGD resulted in two mouth-form regulators. Gene dosage studies revealed a diverged role of these developmental switches, with functional redundancy and quantitative effects in the two mouth-form decisions, respectively. The third morph is cannibalistic and kills kin, whereas the other two morphs do not. Thus, the recent evolution of a new morph relies on pre-existing regulatory mechanisms and adds behavioural and social complexity.

One-Sentence Summary

Experimental genetics in a nematode reveals a key role for developmental plasticity in the evolution of nutritional diversity
optiPRM: A targeted immunopeptidomics LC-MS workflow with ultra-high sensitivity for the detection of mutation-derived tumor neoepitopes from limited input material
Authors: Salek Mogjiborahman , Förster Jonas D. , Becker Jonas P. , Meyer Marten , Charoentong Pornpimol , Lyu Yanhong , Lindner Katharina , Lotsch Catharina , Volkmar Michael , Momburg Frank , Poschke Isabel , Fröhling Stefan , Schmitz Marc , Offringa Rienk , Platten Michael , Jäger Dirk , Zörnig Inka , Riemer Angelika B.
Published: August 2023

Personalized cancer immunotherapies such as vaccines and T cell receptor (TCR)-transgenic T cells rely on the presentation of tumor-specific peptides by human leukocyte antigen (HLA) class I molecules to cytotoxic T cells. Such neoepitopes can for example arise from somatic mutations and their identification is crucial for the rational design of new therapeutic interventions. For their detection by liquid chromatography mass spectrometry (LC-MS), we have developed a parameter optimization workflow to tune targeted assays for maximum detection sensitivity on a per peptide basis, termed optiPRM. Optimization of collision energy using optiPRM allows for improved detection of low abundant peptides that are very hard to detect using standard parameters. Applying this to immunopeptidomics, we detected a neoepitope in a patient-derived xenograft (PDX) from as little as 2.5×106cells input. Application of the workflow on small patient tumor samples allowed for the detection of five mutation-derived neoepitopes in three patients. One neoepitope was confirmed to be recognized by patient T cells. In conclusion, we here present optiPRM, a targeted MS workflow reaching ultra-high sensitivity by per peptide parameter optimization, which allowed for the identification of actionable neoepitopes from sample sizes usually available in the clinic.
Self-supervised learning for characterising histomorphological diversity and spatial RNA expression prediction across 23 human tissue types
Authors: Cisternino Francesco , Ometto Sara , Chatterjee Soumick , Giacopuzzi Edoardo , Levine Adam P. , Glastonbury Craig A.
Published: August 2023

As vast histological archives are digitised, there is a pressing need to be able to associate specific tissue substructures and incident pathology to disease outcomes without arduous annotation. Such automation provides an opportunity to learn fundamental biology about how tissue structure and function varies in a population. Recently, self-supervised learning has proven competitive to supervised machine learning approaches in classification, segmentation and representation learning. Here, we leverage self-supervised learning to generate histology feature representations using 1.7M images across 23 healthy tissues in 838 donors from GTEx. Using these representations, we demonstrate we can automatically segment tissues into their constituent tissue substructures and pathology proportions, and surpass the performance of conventionally used pre-trained models. We observe striking population variability in canonical tissue substructures, highlight examples of missing pathological diagnoses, incorrect assignment of target tissue and cross-tissue contamination. We demonstrate that this variability in tissue composition leads to a likely overestimation of eQTL tissue sharing and drives dramatic differential gene expression changes. We use derived tissue substructures to detect 284 tissue substructures and pathology specific eQTLs. As our derived histology representations are rich morphological descriptors of the underlying tissue, we introduce a multiple instance learning model that can predict and spatially localise individual RNA expression levels directly from histology to specific substructures and pathological features. We validate our RNA spatial predictions with matched ground truth immunohistochemistry (IHC) for several well characterised marker genes, recapitulating their known spatial specificity. Finally, we derive a gene expression spatial enrichment metric, allowing us to detect genes specifically expressed within sites of pathology (e.g. arterial calcification). Together, these results demonstrate the power of self-supervised machine learning when applied to vast histological datasets to allow researchers to pose and answer questions about tissue pathology, its spatial organisation and the interplay between morphological tissue variability and gene expression.
Testing a non-destructive assay to trackPlasmodiumsporozoites in mosquitoes over time
Authors: Oke Catherine E. , Reece Sarah E. , Schneider Petra
Published: August 2023

Background

The extrinsic incubation period (EIP), defined as the time it takes for malaria parasites in a mosquito to become infectious to a vertebrate host, is one of the most influential parameters for malaria transmission but remains poorly understood. The EIP is usually estimated by quantifying salivary gland sporozoites in subsets of mosquitoes, which requires terminal sampling. However, assays that allow repeated sampling of individual mosquitoes over time could provide better resolution of the EIP.

Methods

We tested a non-destructive assay to quantify sporozoites of two rodent malaria species,Plasmodium chabaudiandPlasmodium berghei, expelled throughout 24hr windows, from sugar-feeding substrates using quantitative PCR.

Results

The assay can quantify sporozoites from sugar-feeding substrates, but the prevalence of parasite positive substrates is low. Multiple methods to increase the detection of expelled parasites (running additional technical replicates; using groups rather than individual mosquitoes) did not increase the detection rate, suggesting that expulsion of sporozoites is variable and infrequent.

Conclusions

We reveal successful detection of expelled sporozoites from sugar-feeding substrates. However, investigations of the biological causes underlying the low detection rate of sporozoites (e.g. mosquito feeding behaviour, frequency of sporozoite expulsion, or sporozoite clumping) are needed to maximise the utility of using non-destructive assays to quantify sporozoite dynamics. Increasing detection rates will facilitate the detailed investigation on infection dynamics within mosquitoes, which is necessary to explainPlasmodium’shighly variable EIP and improve understanding of malaria transmission dynamics.
A single-dose intramuscular immunization of pigs with lipid-nanoparticle DNA vaccines based on the hemagglutinin antigen confers complete protection against challenge infection with the homologous influenza virus strain
Authors: Nguyen The Nhu , Kumari Sushmita , Sillman Sarah , Chaudhari Jayeshbhai , Lai Danh Cong , Vu Hiep L.X.
Published: August 2023

Influenza A virus of swine (IAV-S) is highly prevalent and causes significant economic losses to swine producers. Due to the highly variable and rapidly evolving nature of the virus, it is critical to develop a safe and versatile vaccine platform that allows frequent updates of the vaccine immunogens to cope with the emergence of new viral strains. The main objective of this study was to assess the feasibility of using lipid nanoparticles (LNPs) as a nanocarrier to deliver DNA plasmid encoding the viral hemagglutinin (HA) gene in pigs. Intramuscular administration of a single dose of the LNP-DNA vaccines resulted in robust systemic and mucosal in pigs. Importantly, the vaccinated pigs were fully protected against challenge infection with the homologous IAV-S strain, with only one out of 12 vaccinated pigs shedding a low amount of viral genomic RNA in its nasal cavity. No gross or microscopic lesions were observed in the lungs of the vaccinated pigs at necropsy. Thus, the LNP-DNA vaccines are highly effective in protecting pigs against the homologous IAV-S strain and can serve as a promising platform for the rapid development of IAV-S vaccines.

Importance

Influenza A virus of swine (IAV-S) is a significant pathogen of swine. The virus also poses a great public health concern due to its zoonotic potential. Although whole-inactivated virus (WIV) vaccines are available to control IAV-S, their heterologous efficacy is limited due to the substantial genetic/antigenic variation of the viral genome. This study provides compelling evidence demonstrating that lipid nanoparticle encapsulated DNA (LNP-DNA) vaccines induce robust systemic and mucosal immunity and complete protection of pigs against challenge infection with the homologous IAV-S strain. Importantly, the LNP-DNA vaccine approach meets the regulatory requirements to serve as an adaptable platform that can be frequently updated to match the emergence of new IAV-S variants. Thus, this LNP-DNA vaccine strategy exhibits great potential in effectively mitigating the economic impact of IAV-S on the swine industry and the associated public health threat.
Design of Cytotoxic T Cell Epitopes by Machine Learning of Human Degrons
Authors: Truex Nicholas L. , Mohapatra Somesh , Melo Mariane , Rodriguez Jacob , Li Na , Abraham Wuhbet , Sementa Deborah , Touti Faycal , Keskin Derin B. , Wu Catherine J. , Irvine Darrell J. , Gómez-Bombarelli Rafael , Pentelute Bradley L.
Published: August 2023

Antigen processing is critical for producing epitope peptides that are presented by HLA molecules for T cell recognition. Therapeutic vaccines aim to harness these epitopes for priming cytotoxic T cell responses against cancer and pathogens, but insufficient processing often reduces vaccine efficacy through limiting the quantity of epitopes released. Here, we set out to improve antigen processing by harnessing protein degradation signals called degrons from the ubiquitin-proteasome system. We used machine learning to generate a computational model that ascribes a proteasomal degradation score between 0 and 100. Epitope peptides with varying degron activities were synthesized and translocated into cells using nontoxic anthrax proteins: protective antigen (PA) and the N-terminus of lethal factor (LFN). Immunogenicity studies revealed epitope sequences with a low score (<25) show pronounced T-cell activation but epitope sequences with a higher score (>75) provide limited activation. This work sheds light on the sequence–activity relationships between proteasomal degradation and epitope immunogenicity, through conserving the epitope region but varying the flanking sequence. We anticipate that future efforts to incorporate proteasomal degradation signals into vaccine designs will lead to enhanced cytotoxic T cell priming by vaccine therapeutics in clinical settings.
Mycobacterium tuberculosisresponse to cholesterol is integrated with environmental pH and potassium levels via a lipid utilization regulator
Authors: Chen Yue , MacGilvary Nathan J. , Tan Shumin
Published: August 2023

How bacterial response to environmental cues and nutritional sources may be integrated in enabling host colonization is poorly understood. Exploiting a reporter-based screen, we discovered that overexpression ofMycobacterium tuberculosis(Mtb) lipid utilization regulators altered Mtb acidic pH response dampening by low environmental potassium (K+). Transcriptional analyses unveiled amplification of Mtb response to acidic pH in the presence of cholesterol, a major carbon source for Mtb during infection, and vice versa. Strikingly, deletion of the putative lipid regulatormce3Rresulted in loss of augmentation of (i) cholesterol response at acidic pH, and (ii) low [K+] response by cholesterol, with minimal effect on Mtb response to each signal individually. Finally, the Δmce3Rmutant was attenuated for colonization in a murine model that recapitulates lesions with lipid-rich foamy macrophages. These findings reveal critical coordination between bacterial response to environmental and nutritional cues, and establish Mce3R as a crucial integrator of this process.
N4-Hydroxycytidine/Molnupiravir Inhibits RNA-Virus Induced Encephalitis by Producing Mutated Viruses with Reduced Fitness
Authors: Ojha Durbadal , Hill Collin S. , Zhou Shuntai , Evans Alyssa B. , Leung Jacqueline M. , Lewis Christine Schneider , Amblard Franck , Schinazi Raymond F. , Baric Ralph S. , Peterson Karin E. , Swanstrom Ronald
Published: August 2023

A diverse group of RNA viruses including Rabies, Polio, La Crosse, West Nile, Zika, Nipah, Eastern and Western equine encephalitis, Venezuelan equine encephalitis, Japanese encephalitis, and tick-borne encephalitis viruses have the ability to gain access to and replicate in the central nervous system (CNS), causing severe neurological disease. Current treatment for these patients is generally limited to supportive care. To address the need for a generalizable antiviral, we utilized a strategy of mutagenesis to limit virus replication. We evaluated ribavirin (RBV), favipiravir (FAV) andN4-hydroxycytidine (NHC) against La Crosse virus (LACV) which is the primary cause of pediatric arboviral encephalitis cases in North America. NHC was more potent than RBV or FAV in neuronal cells. Oral administration of molnupiravir (MOV), the 5’-isobutyryl prodrug of NHC, decreased neurological disease development by 32% following intraperitoneal (IP) infection of LACV. MOV also reduced disease by 23% when virus was administered intranasally (IN). NHC and MOV produced less fit viruses by incorporating predominantly G-to-A or C-to-U mutations. Furthermore, NHC also inhibited two other orthobunyaviruses, Jamestown Canyon virus and Cache Valley virus. Collectively, these studies indicate that NHC/MOV has therapeutic potential to inhibit virus replication and subsequent neurological disease caused by this neurotropic RNA virus.
Genome-wide assays to characterize rAAV integration into human genomic DNA in vivo
Authors: Prout Jaime , Von Stetina Jessica , Cerqueira Gustavo , Chasse Madison , Chen Huei-Mei , Pla Andrew , Resendes Rachel , Sookiasian Danielle , Tazearslan Cagdas , Wright Jason , Thompson John F.
Published: August 2023

Adeno-associated viral (AAV) vectors are used to treat genetic diseases, expressing therapeutic genes from both extrachromosomal episomes and payloads that integrate into the host genome. Assays were developed to evaluate HR-mediated on-target integration and the potential occurrence of off-target integration. While many studies have addressed elements of these processes, proper characterization requires long-read sequencing to ensure that integrated viral DNA is examined and not the more prevalent episomes. We used Oxford Nanopore to characterize integrated DNA and scan the whole genome for off-target integrations. These assays were applied to cell-based and in vivo models to study vectors that correct phenylketonuria (PKU), caused by loss of phenylalanine hydroxylase (PAH). Administration of the human-specific vector in a humanized-liver mouse xenograft model resulted in stable, nuclease-free integration intoPAH. Because detection of rare integration events in a much larger pool of episomal DNA is subject to artifacts, careful assay validation was required. A long-read, genome-wide assay capable of detecting on- and off-target vector integrations showed no evidence of off-target integration. Artifactual false positive events were below the limit of blank. These data support rAAV as an investigational therapeutic for genetic diseases and reinforce the need for characterization of integration assays to avoid artifacts.
DNA polymerase diversity reveals multiple incursions of Polintons during nematode evolution
Authors: Jeong Dae-Eun , Sundrani Sameer , Hall Richard Nelson , Krupovic Mart , Koonin Eugene V. , Fire Andrew Z.
Published: August 2023

Polintons are dsDNA, virus-like self-synthesizing transposons widely found in eukaryotic genomes. Recent metagenomic discoveries of Polinton-like viruses are consistent with the hypothesis that Polintons invade eukaryotic host genomes through infectious viral particles. Nematode genomes contain multiple copies of Polintons and provide an opportunity to explore the natural distribution and evolution of Polintons during this process. We performed an extensive search of Polintons across nematode genomes, identifying multiple full-length Polinton copies in several species. We provide evidence of both ancient Polinton integrations and recent mobility in strains of the same nematode species. In addition to the major nematode Polinton family, we identified a group of Polintons that are overall closely related to the major family, but encode a distinct protein-primed B family DNA polymerase (pPolB) that is related to homologs from a different group of Polintons present outside of theNematoda. Phylogenetic analyses on the pPolBs support the evolutionary scenarios in which these extrinsic pPolBs that seem to derive from Polinton families present in oomycetes and molluscs replaced the canonical pPolB in subsets of Polintons found in terrestrial and marine nematodes, respectively, suggesting inter-phylum horizontal gene transfers. The pPolBs of the terrestrial nematode and oomycete Polintons share a unique feature, an insertion of a HNH nuclease domain, whereas the pPolBs in the marine nematode Polintons share an insertion of a VSR nuclease domain with marine mollusc pPolBs. We hypothesize that horizontal gene transfer occurs among Polintons from widely different but cohabiting hosts.
cGAS/STING and NLRP3 cooperatively activate CD8+ T cell-mediated anti-tumor immunity in colorectal cancer
Authors: Mowat Courtney , Schiller Daniel , Baker Kristi
Published: August 2023

Colorectal cancer (CRC) is a highly prevalent and deadly disease that is largely refractory to immunotherapy. The only CRC subset that responds to these therapies is characterized by prevalent microsatellite instability (MSI), extensive CD8+ T cell infiltration and high expression of innate immune signaling pathways. Endogenous activation of the cGAS/STING pathway is essential for the CD8+ T cell antitumor response in MSI CRCs, suggesting that activating it in other CRCs could boost immunotherapy response rates. We show that cGAS/STING signaling can be enhanced by costimulation of the NLRP3 inflammasome and that dual stimulation increases CD8+ T cell-mediated antitumor immunity in both MSI and non-MSI CRCs. The ability of NLRP3 to boost cGAS/STING signaling was specific and did not occur with activation of other innate immune pathways such as AIM2 or TLRs. Cooperativity between cGAS/STING and NLRP3 proceeded via a positive feedback loop that was inflammasome-independent and required early crosstalk between the signaling mediators and regulation of their gene expression. Notably, increased cGAS/STING signaling enhanced CD8+ T cell activation when in conjunction with anti-PD1 immunotherapy, suggesting that signaling via NLRP3 could further boost this response and render otherwise resistant CRC susceptible to immunotherapy.

Significance

Innate immune signaling pathways cooperatively regulate CD8+ T cell-mediated antitumor immunity in both hot and cold tumors. In addition to serving as predictive biomarkers, these pathways can be therapeutically targeted to increase response rates to immunotherapy while minimizing undesirable adverse events.
De novodesign of buttressed loops for sculpting protein functions
Authors: Jiang Hanlun , Jude Kevin M. , Wu Kejia , Fallas Jorge , Ueda George , Brunette TJ , Hicks Derrick , Pyles Harley , Yang Aerin , Carter Lauren , Lamb Mila , Li Xinting , Levine Paul M. , Stewart Lance , Garcia K. Christopher , Baker David
Published: August 2023

In natural proteins, structured loops play central roles in molecular recognition, signal transduction and enzyme catalysis. However, because of the intrinsic flexibility and irregularity of loop regions, organizing multiple structured loops at protein functional sites has been very difficult to achieve byde novoprotein design. Here we describe a solution to this problem that generates structured loops buttressed by extensive hydrogen bonding interactions with two neighboring loops and with secondary structure elements. We use this approach to design tandem repeat proteins with buttressed loops ranging from 9 to 14 residues in length. Experimental characterization shows the designs are folded and monodisperse, highly soluble, and thermally stable. Crystal structures are in close agreement with the computational design models, with the loops structured and buttressed by their neighbors as designed. We demonstrate the functionality afforded by loop buttressing by designing and characterizing binders for extended peptides in which the loops form one side of an extended binding pocket. The ability to design multiple structured loops should contribute quite generally to efforts to design new protein functions.
Modeling membrane geometries implicitly in Rosetta
Authors: Woods Hope M. , Leman Julia Koehler , Meiler Jens
Published: August 2023

Interactions between membrane proteins (MPs) and lipid bilayers are critical for many cellular functions. In the Rosetta molecular modeling suite, the implicit membrane energy function is based on a “slab” model, which represent the membrane as a flat bilayer. However, in nature membranes often have a curvature that is important for function and/or stability. Even more prevalent, in structural biology research MPs are reconstituted in model membrane systems such as micelles, bicelles, nanodiscs, or liposomes. Thus, we have modified the existing membrane energy potentials within the RosettaMP framework to allow users to model MPs in different membrane geometries. We show that these modifications can be utilized in core applications within Rosetta such as structure refinement, protein-protein docking, and protein design. For MPs structures found in curved membranes, refining these structures in curved, implicit membranes produces higher quality models with structures closer to experimentally determined structures. For MP systems embedded in multiple membranes, representing both membranes results in more favorable scores compared to only representing one of the membranes. Modeling MPs in geometries mimicking the membrane model system used in structure determination can improve model quality and model discrimination.
A pectin-binding peptide with a structural and signaling role in the assembly of the plant cell wall
Authors: Schoenaers Sébastjen , Lee Hyun Kyung , Gonneau Martine , Faucher Elvina , Levasseur Thomas , Akary Elodie , Claeijs Naomi , Moussu Steven , Broyart Caroline , Balcerowicz Daria , AbdElgawad Hamada , Bassi Andrea , Damineli Daniel Santa Cruz , Costa Alex , Feijó José A , Moreau Celine , Bonnin Estelle , Cathala Bernard , Santiago Julia , Höfte Herman , Vissenberg Kris
Published: August 2023

Pressurized cells with strong walls make up the hydrostatic skeleton of plants. Assembly and expansion of such stressed walls depend on a family of secreted RAPID ALKALINIZATION FACTOR (RALF) peptides which, curiously, bind both a membrane receptor complex and wall-localized LEUCINE-RICH REPEAT EXTENSINs (LRXs) in a mutually exclusive way. Here we show that, in root hairs, the RALF22 peptide has a dual structural and signaling role in cell expansion. Together with LRX1, it directs the compaction of charged pectin polymers at the root hair tip into periodic circumferential rings. Free RALF22 induces the formation of a complex with LORELEI-LIKE-GPI-ANCHORED PROTEIN 1 (LLG1) and FERONIA (FER), triggering adaptive cellular responses. These findings show how a peptide simultaneously functions as a structural component organizing cell wall architecture and as a signaling molecule that regulates this process. This mechanism may also underlie wall assembly and expansion in other plant cell types.