bluuebunny/crossref_metadata_2025 huggingface dataset:
Selective ablation of thymic and peripheral Foxp3+regulatory T cell development
Authors: Yilmazer Acelya , Zevla Dimitra Maria , Malmkvist Rikke , Bello Rodríguez Carlos Alejandro , Undurraga Pablo , Kirgin Emre , Boernert Marie , Voehringer David , Kershaw Olivia , Schlenner Susan , Kretschmer Karsten
Published: August 2023

Foxp3+Treg cells of thymic (tTreg) and peripheral (pTreg) developmental origin are thought to synergistically act to ensure immune homeostasis, with self-reactive tTreg cells primarily constraining autoimmune responses. We exploited tTreg-specific GFP/Cre recombinase activity to selectively ablate either tTreg (ΔtTreg) or pTreg (ΔpTreg) cell development. In contrast to the tTreg cell behavior in ΔpTreg mice, pTreg cells with a highly activated suppressor phenotype replenished the Treg cell pool of C57BL/6.ΔtTreg mice, preventing early mortality and fatal autoimmunity. Even with advancing age, pTreg cells largely maintained immune tolerance in C57BL/6.ΔtTreg mice. However, only two generations of (C57BL/6>NOD) backcrossing precipitated severe disease lethality associated with a distinct, partially overlapping pattern of organ-specific autoimmunity. Genetic association studies defined a small set of autoimmune risk loci sufficient to unleash a particularly severe form of diabetes, including genes known to impinge on Treg cell biology. Thus, pTreg cells exhibit an unexpectedly high functional adaptability, emphasizing their importance as mediators of bystander effects to ensure self-tolerance.

This study in complementary loss-of-function mouse models uncovers an unexpected functional plasticity of pTreg cells in constraining systemic autoimmune responses in the absence of tTreg cells and identifies tTreg cells as primary regulators of β-cell autoimmunity in type 1 diabetes.
Lack of p38 activation in T cells increases IL-35 production and protects against obesity by promoting thermogenesis
Authors: Nikolic Ivana , Ruiz-Garrido Irene , Crespo María , Romero-Becerra Rafael , Leiva-Vega Luis , Mora Alfonso , León Marta , Rodríguez Elena , Leiva Magdalena , Plata-Gómez Ana Belén , Alvarez Maria Beatriz , Torres Jorge L. , Hernández-Cosido Lourdes , López Juan Antonio , Vázquez Jesús , Efeyan Alejo , Martin Pilar , Marcos Miguel , Sabio Guadalupe
Published: August 2023

Obesity is characterized with low grade inflammation, energy imbalance and impaired thermogenesis. The role of regulatory T cells (Treg) in inflammation-mediated maladaptive thermogenesis has not been well established. We discovered that p38 pathway is a key regulator of T cell-mediated adipose tissue (AT) inflammation and browning. Mice with T cells specific deletion of the p38 activators, MKK3/6, were protected against diet-induced obesity and AT inflammation improving their metabolic profile, higher browning and thermogenesis. We identified IL-35 as a driver of adipocyte thermogenic program through ATF2/UCP1/FGF21 pathway. IL-35 limits CD8+T cell infiltration and inflammation in AT. Interestingly, we found that IL35 was reduced in visceral fat from obese patients. Mechanistically we showed that p38 controls the expression of IL-35 in human and mouse Treg cells through mTOR pathway activation. Our findings highlight p38 signaling as a molecular orchestrator of AT T cell accumulation and function and identify p38 and IL-35 as promising targets for metabolic diseases.
Tumor cell-derived lactic acid inhibits the interaction of PD-L1 protein and PD-L1 antibody in the PD-L1/PD-1 blockade therapy-resistant tumor
Authors: Oh Wonkyung , Kim Alyssa Min Jung , Dhawan Deepika , Knapp Deborah W , Lim Seung-Oe
Published: August 2023

Immune checkpoint blockade therapy targeting the PD-1/PD-L1 axis has shown remarkable clinical impact in multiple cancer types. Nontheless, despite the recent success of PD-1/PD-L1 blockade therapy, such response rates in cancer patients have been limited to tumors encompassing specific tumor microenvironment characteristics. The altered metabolic activity of cancer cells shapes the anti-tumor immune response by affecting the activity of immune cells. However, it remains mostly unknown how the altered metabolic activity of cancer cells impacts their resistance to PD-1/PD-L1 blockade therapy. Here we found that tumor cell-derived lactic acid renders the immunosuppressive tumor microenvironment in the PD-1/PD-L1 blockade-resistant tumors by inhibiting the interaction between the PD-L1 protein and anti-PD-L1 antibody. Furthermore, we showed that the combination therapy of targeting PD-L1 with our PD-L1 antibody-drug conjugate (PD-L1-ADC) and reducing lactic acid with the MCT-1 inhibitor, AZD3965, can effectively treat the PD-1/PD-L1 blockade resistant tumors. The findings in this study provide a new mechanism of how lactic acid induces an immunosuppressive environment and suggest a potential combination treatment to overcome the PD-1/PD-L1 blockade therapy resistance.
Allergic inflammation triggers dyslipidemia via IgG signalling
Authors: Fernández-Gallego Nieves , Castillo-González Raquel , Moreno-Serna Lucía , García-Cívico Antonio J. , Sánchez-Martínez Elisa , López-Sanz Celia , Fontes Ana Luiza , Pimentel Lígia L. , Gradillas Ana , Obeso David , Neuhaus René , Ramírez-Huesca Marta , Ruiz-Fernández Ignacio , Nuñez-Borque Emilio , Carrasco Yolanda R. , Ibáñez Borja , Martín Pilar , Blanco Carlos , Barbas Coral , Barber Domingo , Rodríguez-Alcalá Luis M. , Villaseñor Alma , Esteban Vanesa , Sánchez-Madrid Francisco , Jiménez-Saiz Rodrigo
Published: August 2023

Background

Allergic diseases begin early in life and are often chronic, thus creating an inflammatory environment that may precede or exacerbate other pathologies. In this regard, allergy has been associated to metabolic disorders and with a higher risk of cardiovascular disease, but the underlying mechanisms remain incompletely understood.

Methods

We used a murine model of allergy and atherosclerosis, different diets and sensitization methods, and cell-depleting strategies to ascertain the contribution of acute and late phase inflammation to dyslipidemia. Untargeted lipidomic analyses were applied to define the lipid fingerprint of allergic inflammation at different phases of allergic pathology. Expression of genes related to lipid metabolism was assessed in liver and adipose tissue at different times post-allergen challenge. Also, changes in serum triglycerides (TG) were evaluated in a group of 59 patients ≥14 days after the onset of an allergic reaction.

Results

We found that allergic inflammation induces a unique lipid signature that is characterized by increased serum TG and changes in the expression of genes related to lipid metabolism in liver and adipose tissue. Alterations in blood TGs following an allergic reaction are independent of T-cell-driven late phase inflammation. On the contrary, the IgG-mediated alternative pathway of anaphylaxis is sufficient to induce a TG increase and a unique lipid profile. Lastly, we demonstrated an increase in serum TG in 59 patients after undergoing an allergic reaction.

Conclusion

Overall, this study reveals that IgG-mediated allergic inflammation regulates lipid metabolism.
Engineering the highly productive cyanobacteriumSynechococcussp. PCC 11901
Authors: Victoria Angelo J. , Selão Tiago Toscano , Moreno-Cabezuelo José Ángel , Mills Lauren A. , Gale Grant A. R. , Lea-Smith David J. , McCormick Alistair J.
Published: August 2023

Synechococcussp. PCC 11901 (PCC 11901) is a fast-growing marine cyanobacterial strain that has a capacity for sustained biomass accumulation to very high cell densities, comparable to that achieved by commercially relevant heterotrophic organisms. However, genetic tools to engineer PCC 11901 for biotechnology applications are limited. Here we describe a suite of tools based on the CyanoGate MoClo system to unlock the engineering potential of PCC 11901. First, we characterised neutral sites suitable for stable genomic integration that do not affect growth even at high cell densities. Second, we tested a suite of constitutive promoters, terminators, and inducible promoters including a 2,4-diacetylphloroglucinol (DAPG)-inducible PhlF repressor system, which has not previously been demonstrated in cyanobacteria and showed tight regulation and a 228-fold dynamic range of induction. Lastly, we developed a DAPG-inducible dCas9-based CRISPR interference (CRISPRi) system and a modular method to generate markerless mutants using CRISPR-Cas12a. Based on our findings, PCC 11901 is highly responsive to CRISPRi-based repression and showed high efficiencies for single insertion (31-81%) and multiplex double insertion (25%) genome editing with Cas12a. We envision that these tools will lay the foundations for the adoption of PCC 11901 as a robust model strain for engineering biology and green biotechnology.

On sentence summary

Genetic parts were characterised inSynechococcussp. PCC 11901, including a tightly regulated inducible promoter system, efficient CRISPRi and a novel markerless Cas12a genome editing approach.
Loss of Polycomb Repressive Complex 2 function causes asparaginase resistance in T-acute lymphoblastic leukemia through decreased WNT pathway activity
Authors: Lefeivre Thomas , Tudose Cosmin , Grosu Theodora-Ioana , Jones Luke , León Theresa E. , Wynne Kieran , Oliviero Giorgio , Smith Owen P. , Trinquand Amélie , Mansour Marc R. , Ryan Colm J. , Bond Jonathan
Published: August 2023

Loss-of-function mutations and deletions in core components of the epigenetic Polycomb Repressive Complex 2 (PRC2) are associated with poor prognosis and treatment resistance in T-acute lymphoblastic leukemia (T-ALL). We leveraged clinical mutational and transcriptional data to identify a functional link between PRC2 alterations and changes in WNT signaling pathway activity in leukemia cells. Computational integration of transcriptomic, proteomic and phosphoproteomic data from an isogenic T-ALL cellular model revealed reduced activity of the WNT-dependent stabilization of proteins (WNT/STOP) pathway in cells lacking core PRC2 factor EZH2. We discovered that PRC2 loss significantly reduced sensitivity to key T-ALL treatment asparaginase, and that this was mechanistically linked to increased cellular ubiquitination levels that bolstered leukemia cell asparagine reserves. We further found that asparaginase resistance in PRC2-depleted leukemic blasts could be mitigated by pharmaceutical proteasome inhibition, thereby providing a novel and clinically tractable means to tackle induction treatment failure in high-risk T-ALL.
A genetic basis for cancer sex differences revealed in Xp11 translocation renal cell carcinoma
Authors: Achom Mingkee , Sadagopan Ananthan , Bao Chunyang , McBride Fiona , Xu Qingru , Konda Prathyusha , Tourdot Richard W. , Li Jiao , Nakhoul Maria , Gallant Daniel S. , Ahmed Usman Ali , O’Toole Jillian , Freeman Dory , Lee Gwo-Shu Mary , Hecht Jonathan L. , Kauffman Eric C. , Einstein David J , Choueiri Toni K. , Zhang Cheng-Zhong , Viswanathan Srinivas R.
Published: August 2023

Xp11 translocation renal cell carcinoma (tRCC) is a female-predominant kidney cancer driven by translocations between theTFE3gene on chromosome Xp11.2 and partner genes located on either chrX or on autosomes. The rearrangement processes that underlieTFE3fusions, and whether they are linked to the female sex bias of this cancer, are largely unexplored. Moreover, whether oncogenicTFE3fusions arise from both the active and inactive X chromosomes in females remains unknown. Here we address these questions by haplotype-specific analyses of whole-genome sequences of 29 tRCC samples from 15 patients and by re-analysis of 145 published tRCC whole-exome sequences. We show thatTFE3fusions universally arise as reciprocal translocations with minimal DNA loss or insertion at paired break ends. Strikingly, we observe a near exact 2:1 female:male ratio inTFE3fusions arising via X:autosomal translocation (but not via X inversion), which accounts for the female predominance of tRCC. This 2:1 ratio is at least partially attributable to oncogenic fusions involving the inactive X chromosome and is accompanied by partial re-activation of silenced chrX genes on the rearranged chromosome. Our results highlight how somatic alterations involving the X chromosome place unique constraints on tumor initiation and exemplify how genetic rearrangements of the sex chromosomes can underlie cancer sex differences.
FLN-2 functions in parallel to LINC complexes and Cdc42/actin pathways during P-cell nuclear migration through constricted spaces inCaenorhabditis elegans
Authors: Ma Linda , Kuhn Jonathan , Chang Yu-Tai , Elnatan Daniel , Luxton G.W. Gant , Starr Daniel A.
Published: August 2023

Nuclear migration through narrow constrictions is important for development, metastasis, and pro-inflammatory responses. Studies performed in tissue culture cells have implicated LINC (linker of nucleoskeleton and cytoskeleton) complexes, microtubule motors, the actin cytoskeleton, and nuclear envelope repair machinery as important mediators of nuclear movements through constricted spaces. However, little is understood about how these mechanisms operate to move nucleiin vivo. InC. eleganslarvae, 6 pairs of hypodermal P cells migrate from lateral to ventral positions through a constricted space between the body wall muscles and the cuticle. P-cell nuclear migration is mediated in part by LINC complexes using a microtubule-based pathway and by an independent CDC-42/actin-based pathway. However, when both LINC complex and actin-based pathways are knocked out, many nuclei still migrate, suggesting the existence of additional pathways. Here we show that FLN-2 functions in a third pathway to mediate P-cell nuclear migration. The predicted N-terminal actin binding domain in FLN-2 that is found in canonical filamins is dispensable for FLN-2 function, this and structural predictions suggest that FLN-2 is not a divergent filamin. The immunoglobulin (Ig)-like repeats 4-8 of FLN-2 were necessary for P-cell nuclear migration. Furthermore, in the absence of the LINC complex componentunc-84,fln-2mutants had an increase in P-cell nuclear rupture. We conclude that FLN-2 functions to maintain the integrity of the nuclear envelope in parallel with the LINC complex and CDC-42/actin-based pathways to move P-cell nuclei through constricted spaces.
Monarch butterflyCryptochrome 1loss-of-function mutants reveal differences in light entrainment of 24-hour behavioral rhythms in insects
Authors: Iiams Samantha E. , Wan Guijun , Zhang Jiwei , Lugena Aldrin B. , Zhang Ying , Hayden Ashley N. , Merlin Christine
Published: August 2023

Light is one of the strongest cues for entrainment of circadian clocks in most organisms. Previous work inDrosophila melanogaster(dm) has shown that entrainment relies on both the visual system and the circadian, blue-light photoreceptor Cryptochrome (dmCRY). Here, we used the monarch butterflyDanaus plexippus(dp) to test conservation of this mechanism among insects and the relative importance of monarchCryptochrome 1(dpCry1) in the entrainment of its clockin vivo. We showed that loss of functionaldpCry1abolishes adult circadian eclosion behavior and molecular circadian rhythms in the monarch brain. These rhythms can be restored by entrainment to temperature cycles, demonstrating that the core circadian clock is intact indpCry1mutants. Importantly, we showed that rhythmic flight activity is also disrupted indpCry1mutants but not in the visually impaireddpNinaB1mutants, suggesting that unlikeDrosophilalight-entrainment of the monarch circadian clock relies solely on dpCRY1 photoreception.
Characterization and contribution of RPE senescence to Age-related macular degeneration inTnfrsf10knock out mice
Authors: Wada Iori , Mori Kenichiro , Sreekumar Parameswaran G , Ji Rui , Spee Christine , Hong Elise , Ishikawa Keijiro , Sonoda Koh-Hei , Kannan Ram
Published: August 2023

Background

Retinal pigment epithelial cells (RPE) play vital role in the pathogenesis of age-related macular degeneration (AMD). Our laboratory has shown that RPE cellular senescence contributed to the pathophysiology of experimental AMD, and SASP members are involved in this process. Recently, we presented confirmatory evidence to earlier GWAS studies that dysregulation of tumor necrosis factor receptor superfamily 10A (TNFRSF10A) dysregulation leads to AMD development and is linked to RPE dysfunction. This study aims to investigate the contribution of RPE senescence to AMD pathophysiology usingTNFRSF10Asilenced human RPE (hRPE) cells and Tnfrsf10 KO mice.

Methods

Sub-confluent primary hRPE cells andTNFRSF10Asilenced hRPE were exposed to stress-induced premature senescence with H2O2 (500 μM, 48h), and senescence-associated markers (βgal, p16, and p21) were analyzed by RT-PCR and WB analysis. The effect of H2O2-induced senescence in non-silenced and silenced hRPE on OXPHOS and glycolysis was determined using Seahorse XF96 analyzer. Male C57BL/6J Tnfrsf10 KO (Tnfrsf10-/-) mice were used to study the regulation of senescence by TNFRSF10Ain vivo. Expression of p16 and p21 in control and KO mice of varying ages were determined by RT-PCR, WB, and immunostaining analysis.

Results

The senescence-associated p16 and p21 showed a significant (p< 0.01) upregulation with H2O2 induction at the gene (1.8- and 3-fold) and protein (3.2- and 4-fold) levels in hRPE cells. The protein expression of p16 and p21 was further significantly increased by co-treatment with siRNA (p< 0.05 vs. H2O2). Mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) (pmol/min/total DNA) increased with senescence induction by H2O2 for 48h in control RPE, and knockdown ofTNFRSF10Acaused a further increase in OCR and ECAR. In addition, co-treatment with PKC activator significantly improved all parameters. Similarly,in vivostudies showed upregulation of p16 and p21 by RT-PCR, WB, and immunostaining analysis in RPE/choroid of Tnfrsf10 KO mice. When subjected to examination across distinct age groups, namely young (1-3 months), middle (6-9 months), and old (12-15 months) mice, a discernible age-related elevation in the expression of p16 and p21 was observed.

Conclusions

Our findings suggest that TNRSF10A is a regulator of regulates in RPE senescence. Further work on elucidating pathways of senescence will facilitate the development of new therapeutic targets for AMD.
A genome-wide cytotoxicity screen of Cluster F1 mycobacteriophage Girr reveals novel inhibitors ofMycobacterium smegmatisgrowth
Authors: Pollenz Richard S , Barnhill Kaylee , Biggs Abbigail , Bland Jackson , Carter Victoria , Chase Michael , Clark Hayley , Coleman Caitlyn , Daffner Marshall , Deam Caitlyn , Finocchiaro Alyssa , Franco Vanessa , Fuller Thomas , Pinera Juan Gallardo , Horne Mae , Howard Zoe , Kanahan Olivia , Miklaszewski Christopher , Miller Sydney , Morgan Ryan , Onalaja Oluwatobi , Otero Louis , Padhye Shivani , Rainey Emily , Rasul Fareed , Rodier Alexandra , Schlosser Sydni , Sciacchitano Ava , Stewart Emma , Thakkar Rajvi , Heller Danielle
Published: August 2023

Over the past decade, thousands of bacteriophage genomes have been sequenced and annotated. A striking observation from this work is that known structural features and functions cannot be assigned for >65% of the encoded proteins. One approach to begin experimentally elucidating the function of these uncharacterized gene products is genome-wide screening to identify phage genes that confer phenotypes of interest like inhibition of host growth. This study describes the results of a screen evaluating the effects of overexpressing each gene encoded by the temperate Cluster F1 mycobacteriophage Girr on the growth of the host bacteriumMycobacterium smegmatis. Overexpression of 29 of the 102 Girr genes (∼28% of the genome) resulted in mild to severe cytotoxicity. Of the 29 toxic genes described, 12 have no known function (NKF) and are predominately small proteins of <125 amino acids. Overexpression of the majority of these 12 cytotoxic NKF proteins resulted in moderate to severe growth reduction and represent novel antimicrobial products. The remaining 17 toxic genes have predicted functions, encoding products involved in phage structure, DNA replication/modification, DNA binding/gene regulation, or other enzymatic activity. Comparison of this dataset with prior genome-wide cytotoxicity screens of mycobacteriophages Waterfoul and Hammy reveals some common functional themes, though several of the predicted Girr functions associated with cytotoxicity in our report, including genes involved in lysogeny, have not been described previously. This study, completed as part of the HHMI-supported SEA-GENES project, highlights the power of parallel, genome-wide overexpression screens to identify novel interactions between phages and their hosts.
MEK inhibition enhances the antitumor effect of radiation therapy inNF1-deficient glioblastoma
Authors: Ioannou Maria , Lalwani Kriti , Ayanlaja Abiola A. , Chinnasamy Viveka , Pratilas Christine A. , Schreck Karisa C.
Published: August 2023

Individuals with neurofibromatosis type 1 (NF-1), an autosomal dominant neurogenetic and tumor predisposition syndrome, are susceptible to developing low-grade glioma (LGG) and, less commonly, high-grade glioma (HGG). These gliomas exhibit loss of the neurofibromin gene (NF1), and 10-15% of sporadic HGG have somaticNF1alterations. Loss of NF1 leads to hyperactive RAS signaling, creating opportunity given the established efficacy of MEK inhibitors (MEKi) in plexiform neurofibromas and some individuals with LGG. We observed thatNF1-deficient glioblastoma neurospheres were sensitive to the combination of a MEKi (mirdametinib) with irradiation, as evidenced by synergistic inhibition of cell growth, colony formation, and increased cell death. In contrast,NF1-intact neurospheres were not sensitive to the combination, despite complete ERK pathway inhibition. No neurosphere lines exhibited enhanced sensitivity to temozolomide combined with mirdametinib. Mirdametinib decreased transcription of homologous recombination genes and RAD51 foci, associated with DNA damage repair, in sensitive models. Heterotopic xenograft models displayed synergistic growth inhibition to mirdametinib combined with irradiation inNF1-deficient glioma xenografts, but not those with intactNF1. In sensitive models, benefits were observed at least three weeks beyond the completion of treatment, including sustained phospho-ERK inhibition on immunoblot and decreased Ki-67 expression. These observations demonstrate synergistic activity between mirdametinib and irradiation inNF1-deficient glioma models and may have clinical implications for patients with gliomas that harbor germline or somaticNF1alterations.
Colchicine inhibited oxidative stress-induced endothelial cell senescence via blocking NF-κB and MAPKs: Implications in vascular diseases
Authors: Zhou Huakang , Khan Dilaware , Hussain Sajid Muhammad , Gerdes Norbert , Hagenbeck Carsten , Rana Majeed , Cornelius Jan Frederick , Muhammad Sajjad
Published: August 2023

Smoking, alcohol abuse, and hypertension are – among others the potential risk factors for cardiovascular diseases. These risk factors generate oxidative stress and cause oxidative stress-induced DNA damage, resulting in cellular senescence and senescence-associated secretory phenotype (SASP). The SASP factors in feed-forward response exacerbate inflammation and cause tissue remodeling, resulting in atherosclerotic plaque formation and rupture. Colchicine was used to ameliorate oxidative stress-induced senescence in human umbilical vein endothelial cells. Oxidative stress was quantified by reactive oxygen species (ROS) assay and oxidative stress-induced DNA damage was analyzed by 8-OHDG immunofluorescence staining. Endothelial cell senescence was visualized by β-gal staining. The relative mRNA expression was quantified by qPCR and protein analysis was performed by Western blot. Colchicine inhibited ROS generation and mitigated oxidative stress-induced DNA damage. It dampened oxidative stress-induced endothelial cell senescence and improved the expression of DNA repair protein KU80 and aging marker Lamin B1. The drug attenuated the expression of senescence marker P21 at mRNA and protein levels. The pathway analysis showed that colchicine inhibited NF-κB and MAPKs pathways and subdued mTOR activation. Colchicine also attenuated mRNA expression of interleukin (IL)-1β, IL-6, IL-8 MCP-1, ICAM-1, and E-selectin. Furthermore, colchicine reduced the mRNA and protein expression of matrix metalloproteinase (MMP-2). In summary, colchicine blocked oxidative stress-induced senescence and SASP by inhibiting the activation of NF-κB and MAPKs pathways.
Comparative Analysis of Molecular Pathogenic Mechanisms and Antiviral Development Targeting Old and New World Hantaviruses
Authors: Jeyachandran Arjit Vijey , Irudayam Joseph Ignatius , Dubey Swati , Chakravarty Nikhil , Konda Bindu , Shah Aayushi , Su Baolong , Wang Cheng , Cui Qi , Williams Kevin J. , Srikanth Sonal , Shi Yanhong , Deb Arjun , Damoiseaux Robert , Stripp Barry R. , Ramaiah Arunachalam , Arumugaswami Vaithilingaraja
Published: August 2023

Background

Hantaviruses – dichotomized into New World (i.e. Andes virus, ANDV; Sin Nombre virus, SNV) and Old-World viruses (i.e. Hantaan virus, HTNV) – are zoonotic viruses transmitted from rodents to humans. Currently, no FDA-approved vaccines against hantaviruses exist. Given the recent breakthrough to human-human transmission by the ANDV, an essential step is to establish an effective pandemic preparedness infrastructure to rapidly identify cell tropism, infective potential, and effective therapeutic agents through systematic investigation.

Methods

We established human cell model systems in lung (airway and distal lung epithelial cells), heart (pluripotent stem cell-derived (PSC-) cardiomyocytes), and brain (PSC-astrocytes) cell types and subsequently evaluated ANDV, HTNV and SNV tropisms. Transcriptomic, lipidomic and bioinformatic data analyses were performed to identify the molecular pathogenic mechanisms of viruses in different cell types. This cell-based infection system was utilized to establish a drug testing platform and pharmacogenomic comparisons.

Results

ANDV showed broad tropism for all cell types assessed. HTNV replication was predominantly observed in heart and brain cells. ANDV efficiently replicated in human and mouse 3D distal lung organoids. Transcriptomic analysis showed that ANDV infection resulted in pronounced inflammatory response and downregulation of cholesterol biosynthesis pathway in lung cells. Lipidomic profiling revealed that ANDV-infected cells showed reduced level of cholesterol esters and triglycerides. Further analysis of pathway-based molecular signatures showed that, compared to SNV and HTNV, ANDV infection caused drastic lung cell injury responses. A selective drug screening identified STING agonists, nucleoside analogues and plant-derived compounds that inhibited ANDV viral infection and rescued cellular metabolism. In line with experimental results, transcriptome data shows that the least number of total and unique differentially expressed genes were identified in urolithin B- and favipiravir-treated cells, confirming the higher efficiency of these two drugs in inhibiting ANDV, resulting in host cell ability to balance gene expression to establish proper cell functioning.

Conclusions

Overall, our study describes advanced human PSC-derived model systems and systems-level transcriptomics and lipidomic data to better understand Old and New World hantaviral tropism, as well as drug candidates that can be further assessed for potential rapid deployment in the event of a pandemic.
Nitrite secretion by cyanobacteria is controlled by the small protein NirP1
Authors: Kraus Alexander , Spät Philipp , Timm Stefan , Wilson Amy , Schumann Rhena , Hagemann Martin , Macek Boris , Hess Wolfgang R.
Published: August 2023

When the supply of inorganic carbon is limiting, photosynthetic cyanobacteria excrete nitrite, a toxic intermediate in the ammonia assimilation pathway from nitrate. While it has been hypothesized that the secreted nitrite represents excess nitrogen that could not be further assimilated due to the missing carbon, the underlying molecular mechanism has remained enigmatic. Here, we characterized a hitherto unannotated gene with homologs in the genomes of 485 different cyanobacteria that is upregulated under low carbon conditions and controlled by the transcription factor NtcA, a central regulator of nitrogen homeostasis. To understand its function, we ectopically overexpressed it inSynechocystissp. PCC 6803, which resulted in a chlorotic phenotype, delayed growth, severe changes in amino acid pools, and nitrite excretion. Coimmunoprecipitation experiments revealed that this protein targets nitrite reductase, a central enzyme in the assimilation of ammonia from nitrate/nitrite, and was re-named to nitrite reductase regulator protein 1 (NirP1). Our results reveal that NirP1 is widely conserved in cyanobacteria and plays a crucial role in the coordination of C/N primary metabolism by targeting one of the central enzymes. In natural environments, the excreted nitrite will be utilized by other microorganisms; therefore, NirP1 ultimately impacts the activities and composition of the surrounding microbiome.
PEX1 is essential for the glycosome biogenesis and trypanosomatid parasite survival
Authors: Mahadevan Lavanya , Arya Hemant , Schliebs Wolfgang , Erdmann Ralf , Kalel Vishal C.
Published: August 2023

Trypanosomatid parasites are kinetoplastid protists that compartmentalize glycolytic enzymes in unique peroxisome-related organelles called glycosomes. The heterohexameric AAA-ATPase complex of PEX1-PEX6 is anchored to the peroxisomal membrane and functions in the export of matrix protein import receptor PEX5 from the peroxisomal membrane. Defects in PEX1, PEX6 or their membrane anchor causes dysfunction of peroxisomal matrix protein import cycle. In this study, we identified theTrypanosomaPEX1 orthologue using sequence and structural similarities. Using yeast two-hybrid analysis, we demonstrate thatTbPEX1 can bind toTbPEX6. Endogenously taggedTbPEX1 localizes to glycosomes in theT. bruceiparasites. Depletion of PEX1 gene expression by RNA interference causes lethality to bloodstream form trypanosomes, due to a partial mislocalization of glycosomal enzymes to the cytosol and ATP depletion.TbPEX1 RNAi leads to a selective proteasomal degradation of both matrix protein import receptorsTbPEX5 andTbPEX7. Unlike in yeast, PEX1 depletion did not result in an accumulation of ubiquitinatedTbPEX5 in trypanosomes. As PEX1 turned out to be essential for trypanosomatid parasites, it could provide a suitable drug target for parasitic diseases. The results also suggests that these parasites possess a highly efficient quality control mechanisms that export the import receptors from glycosomes to the cytosol, in the absence of a functionalTbPEX1-TbPEX6 complex.
Postzygotic barriers persist despite ongoing introgression in hybridizingMimulusspecies
Authors: Mantel Samuel J. , Sweigart Andrea L.
Published: August 2023

The evolution of postzygotic isolation is thought to be a key step in maintaining species boundaries upon secondary contact, yet the dynamics and persistence of hybrid incompatibilities in sympatric species are not well understood.

Here, we explore these issues using genetic mapping in three populations of recombinant inbred lines between naturally hybridizing monkeyflowersMimulus guttatusandM. nasutusfrom the sympatric Catherine Creek population.

The threeM. guttatusfounders differ dramatically in admixture history. Comparative genetic mapping also reveals three putative inversions segregating among theM. guttatusfounders, two due to admixture. We observe strong, genome-wide transmission ratio distortion, but patterns are highly variable among populations. Some distortion is explained by epistatic selection favoring parental genotypes, but tests of inter-chromosomal linkage disequilibrium also reveal multiple candidate Dobzhansky-Muller incompatibilities. We also map several genetic loci for hybrid fertility, including two interacting pairs coinciding with peaks of distortion.

Remarkably, in this limited sample ofM. guttatus, we discover abundant segregating variation for hybrid incompatibilities withM. nasutus,suggesting this population harbors diverse contributors to postzygotic isolation. Moreover, even with substantial admixture, hybrid incompatibilities betweenMimulusspecies persist, suggesting postzygotic isolation might be a potent force in maintaining species barriers in this system.
Multiplexed amplicon sequencing reveals the heterogeneous spatial distribution of pyrethroid resistance mutations inAedes albopictusmosquito populations in Southern France
Authors: Fontaine Albin , Mignotte Antoine , Lacour Guillaume , Nguyen Agnès , Gomez Nicolas , Chanaud Lionel , L’Ambert Grégory , Briolant Sébastien
Published: August 2023

The risk of mosquito-borne diseases transmission is moving fast toward temperate climates with the colonization and proliferation of the Asian tiger mosquito vectorAedes albopictusand the rapid and mass transport of passengers returning from tropical regions where the viruses are endemic. The prevention of majorAedes-borne viruses heavily relies on the use of insecticides for vector control, mainly pyrethroids In Europe. High-throughput molecular assays can provide a cost-effective surrogate to phenotypic insecticide resistance assays when mutations have been previously linked to a resistance phenotype. Here, we screened for the spatial distribution ofkdrmutations at a large scale using a two-step approach based on multiplexed amplicon sequencing and an unprecedented collection of field-derived mosquitoes in South of France. We identified the presence of the V1016G allele in 14 sites. The V1016G allele was predominantly found in South-East France close to the Italian border with two additional isolated sites close to Bordeaux and Marmande. All mosquitoes were heterozygous for this mutation and should not be phenotypically resistant to pyrethroid insecticide. Four other mutations were identified in our targeted genomic sequence: I1532T, M1006L, M1586L, M995L. Sequencing a section of maternally inherited mitochondrial genome confirmed that the spread ofAe. albopictusin France originated from founders with haplogroup A1. These findings contribute to the broader understanding of resistance dynamics in Europe and can inform targeted approaches to mitigate the impact of resistance on vector control.
Determining the shelf life of an erythropoietin alfa biosimilar GBPD002 through stability study
Authors: Nag Kakon , Mohiuddin Mohammad , Kumar Samir , Khan Md. Maksudur Rahman , Sarker Md. Enamul Haq , Biswas Bipul Kumar , Roy Rony , Molla Md. Tarek , Roy Ratan , Bappi Md. Emrul Hasan , Rahman Arifur , Haq Sheik Rejaul , Sarker Md. Shofiquzzaman , Popy Priyanka Mollik , Mumu Raisa Ferdaushi , Barman Uttam , Oli Md. Shamsul Kaunain , Khoka Md. Sadek Hosen , Sarker Sourav , Alam Md. Firoz , Sultana Naznin
Published: August 2023

GBPD002 is a recombinant human erythropoietin (rhEPO) produced by recombinant DNA technology using mammalian cell expression system. In this study, samples were analyzed according to current Good Laboratory Practice (cGLP) and regulatory guidelines to evaluate the quality of the product under the influence of variety of environmental factors in a time-dependent manner. Accelerated (25 ± 2 °C and relative humidity: 60 ± 5 %) and real-time (5 ± 3 °C) stability study were conducted up to 6 months and 12 months, respectively; samples were analyzed in every 3 months. After 12 months, real-time stability studies were performed with 6 months interval up to18 months, which to be continued up to 24 months. Appearance and pH were assessed using standard methods, and molecular weight was determined by Western blotting. Chromatographic identification and quantitative assays were performed by reversed- phase chromatography (RPC). High-molecular weight aggregatesand degradants were determined using size exclusion chromatography (SEC) and particle size distribution (PSD) analysis. Biofunctionality of the samples were evaluated byin vitroandin vivobioassays. Bacterial endotoxin and sterility test were performed as safety parameters. All samples met the acceptance criteria, and the data were extrapolated using the regulatory guideline to determine the shelf life. The data revealed that the GBPD002 is stable up to 24 months.
Transcriptional control of compartmental boundary positioning during Drosophila wing development
Authors: Aguilar Gustavo , Sickmann Michèle , Bieli Dimitri , Born Gordian , Affolter Markus , Müller Martin
Published: August 2023

The establishment of tissue axes is fundamental during embryonic development. In the Drosophila wing, the anterior/posterior (AP) and the dorsal/ventral (DV) compartment boundaries provide the basic coordinates around which the tissue develops. These boundaries arise as a result of two lineage decisions, the acquisition of posterior fate by the selector geneengrailed(en)and of dorsal fate by the selector geneapterous(ap). Whileenexpression domain is set up during embryogenesis,apexpression only starts during early wing development. Thus, the correct establishment ofapexpression pattern with respect toenmust be tightly controlled. Here we have functionally investigated the transcriptional inputs integrated by the “early”apenhancer (apE) and their requirement for correct boundary positioning. Detailed mutational analyses using CRISPR/Cas revealed a role of apE in positioning the DV boundary with respect to the AP boundary, with apE mutants often displaying mirror-image anterior wing duplications. We then accomplished tissue-specific enhancer disruption via dCas9 expression. This approach allowed us to dissect the spatio-temporal requirement of apE function, challenging the mechanism by which apE miss-regulation leads to AP defects. Base-pair resolution analyses of apE uncovered a single HOX binding site essential for wing development, which, when mutated, led to wingless flies. Along these lines, we found that the HOX gene Antennapedia (Antp) is fundamental forapexpression. In addition, we demonstrated that the transcription factors Pointed (Pnt), Homothorax (Hth) and Grain (Grn) are necessary for apE function. Together, our results provide a comprehensive molecular basis of earlyapactivation and the developmental consequences of its miss-regulation, shedding light on how compartmental boundaries are be set up during development.
Molecularly imprinted nanoparticles reveal regulatory scaffolding features in Pyk2 tyrosine kinase
Authors: Zanela Tania M. Palhano , Zangiabadi Milad , Zhao Yan , Underbakke Eric S.
Published: August 2023

Pyk2 is a multi-domain non-receptor tyrosine kinase that serves dual roles as signaling enzyme and scaffold. Pyk2 activation involves a multi-stage cascade of conformational rearrangements and protein interactions initiated by autophosphorylation of a linker site. Linker phosphorylation recruits Src kinase, and Src-mediated phosphorylation of the Pyk2 activation loop confers full activation. The regulation and accessibility of the initial Pyk2 autophosphorylation site remains unclear. We employed peptide-binding molecularly imprinted nanoparticles (MINPs) to probe the regulatory conformations controlling Pyk2 activation. MINPs differentiating local structure and phosphorylation state revealed that the Pyk2 autophosphorylation site is protected in the autoinhibited state. Activity profiling of Pyk2 variants implicated FERM and linker residues responsible for constraining the autophosphorylation site. MINPs targeting each Src docking site disrupt the higher-order kinase interactions critical for activation complex maturation. Ultimately, MINPs targeting key regulatory motifs establish a useful toolkit for probing successive activational stages in the higher-order Pyk2 signaling complex.

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Topological stress triggers persistent DNA lesions in ribosomal DNA with ensuing formation of PML-nucleolar compartment
Authors: Urbancokova Alexandra , Hornofova Terezie , Novak Josef , Salajkova Sarka Andrs , Stemberkova Hubackova Sona , Uvizl Alena , Buchtova Tereza , Mistrik Martin , Mac Stay Brian , Hodny Zdenek , Bartek Jiri , Vasicova Pavla
Published: August 2023

PML, a multifunctional protein, is crucial for forming PML-nuclear bodies involved in stress responses. Under specific conditions, PML associates with nucleolar caps formed after RNA polymerase I (RNAPI) inhibition, leading to PML-nucleolar associations (PNAs). This study investigates PNAs-inducing stimuli by exposing cells to various genotoxic stresses. We found that the most potent inducers of PNAs introduced topological stress and inhibited RNAPI. Doxorubicin, the most effective compound, induced double-strand breaks (DSBs) in the rDNA locus. PNAs co-localized with damaged rDNA, segregating it from active nucleoli. Cleaving the rDNA locus with I-PpoI confirmed rDNA damage as a genuine stimulus for PNAs. Inhibition of ATM, ATR kinases, and RAD51 reduced I-PpoI-induced PNAs, highlighting the importance of ATM/ATR-dependent nucleolar cap formation and homologous recombination (HR) in their triggering. I-PpoI-induced PNAs co-localized with rDNA DSBs positive for RPA32-pS33 but deficient in RAD51, indicating resected DNA unable to complete HR repair. Our findings suggest that PNAs form in response to persistent rDNA damage within the nucleolar cap, highlighting the interplay between PML/PNAs and rDNA alterations due to topological stress, RNAPI inhibition, and rDNA DSBs destined for HR. Cells with persistent PNAs undergo senescence, suggesting PNAs help avoid rDNA instability, with implications for tumorigenesis and aging.
Accelerated social representational drift in the nucleus accumbens in a model of autism
Authors: Zhao Pingping , Chen Xing , Bellafard Arash , Murugesan Avaneesh , Quan Jonathan , Aharoni Daniel , Golshani Peyman
Published: August 2023

Impaired social interaction is one of the core deficits of autism spectrum disorder (ASD) and may result from social interactions being less rewarding. How the nucleus accumbens (NAc), as a key hub of reward circuitry, encodes social interaction and whether these representations are altered in ASD remain poorly understood. We identified NAc ensembles encoding social interactions by calcium imaging using miniaturized microscopy. NAc population activity, specifically D1 receptor-expressing medium spiny neurons (D1-MSNs) activity, predicted social interaction epochs. Despite a high turnover of NAc neurons modulated by social interaction, we found a stable population code for social interaction in NAc which was dramatically degraded in Cntnap2-/-mouse model of ASD. Surprisingly, non-specific optogenetic inhibition of NAc core neurons increased social interaction time and significantly improved sociability in Cntnap2-/-mice. Inhibition of D1- or D2-MSNs showed reciprocal effects, with D1 inhibition decreasing social interaction and D2 inhibition increasing interaction. Therefore, social interactions are preferentially, specifically and dynamically encoded by NAc neurons and social representations are degraded in this autism model.
Exploiting Activation and Inactivation Mechanisms in Type I-C CRISPR-Cas3 for Genome Editing Applications
Authors: Hu Chunyi , Myers Mason T. , Zhou Xufei , hou Zhonggang , Lozen Macy L. , Zhang Yan , Ke Ailong
Published: August 2023

Type I CRISPR-Cas systems utilize the RNA-guided Cascade complex to identify matching DNA targets, and the nuclease-helicase Cas3 to degrade them. Among seven subtypes, Type I-C is compact in size and highly active in creating large-sized genome deletions in human cells. Here we use four cryo-electron microscopy snapshots to define its RNA-guided DNA binding and cleavage mechanisms in high resolution. The non-target DNA strand (NTS) is accommodated by I-C Cascade in a continuous binding groove along the juxtaposed Cas11 subunits. Binding of Cas3 further traps a flexible bulge in NTS, enabling efficient NTS nicking. We identified two anti-CRISPR proteins AcrIC8 and AcrIC9, that strongly inhibitN. lactamicaI-C function. Structural analysis showed that AcrIC8 inhibits PAM recognition through direct competition, whereas AcrIC9 achieves so through allosteric inhibition. Both Acrs potently inhibit I-C-mediated genome editing and transcriptional modulation in human cells, providing the first off-switches for controllable Type I CRISPR genome engineering.
CRISPR-DIPOFF: An Interpretable Deep Learning Approach for CRISPR Cas-9 Off-Target Prediction
Authors: Toufikuzzaman Md. , Samee Md. Abul Hassan , Rahman M Sohel
Published: August 2023

CRISPR Cas-9 is a groundbreaking gene-editing tool that harnesses bacterial defense systems to alter DNA sequences accurately. This innovative technology holds vast promise in multiple domains like biotechnology, agriculture, and medicine. However, such power does not come without its own peril, and one such issue is the potential for unintended modifications (Off-Target), which highlights the need for accurate prediction and mitigation strategies. Though previous studies have demonstrated improvement in Off-Target prediction capability with the application of deep learning, they often struggle with the precision-recall trade-off, limiting their effectiveness and do not provide proper interpretation of the complex decision-making process of their models. To address these limitations, we have thoroughly explored deep learning networks, particularly the recurrent neural network (RNN) and transformer based models, leveraging their established success in handling sequence data. Furthermore, we have employed genetic algorithm for hyperparameter tuning to optimize these models’ performance. The results from our experiments demonstrate significant performance improvement compared to the current state-of-the-art in Off-Target prediction, highlighting the efficacy of our approach. Furthermore, leveraging the power of the integrated gradient method, we make an effort to interpret our models resulting in a detailed analysis and understanding of the underlying factors that contribute to Off-Target predictions, in particular the presence of two sub-regions in the seed region of sgRNA which extends the established biological hypothesis of Off-Target effects. To the best of our knowledge, our model can be considered as the first model combining high efficacy, interpretability, and a desirable balance between precision and recall.
Enterovirus-A71 exploits Rab11 to recruit chaperones for virus morphogenesis
Authors: Yong Ng Qing , Mahendran Vikneswari , Lim Ze Qin , Chu Justin Jang Hann , Chow Vincent Tk , Sze Newman Siu Kwan , Alonso Sylvie
Published: August 2023

Enterovirus 71 (EV-A71) causes Hand, Foot and Mouth Disease (HFMD) in children and has been associated with neurological complications. A siRNA screen in EV-A71 infected-motor neurons identified small GTPase Rab11a as a pro-viral host factor. Rab11a and Rab11b isoforms were interchangeably exploited by strains from major EV-A71 genogroups and Coxsackievirus 16, another major causative agent of HFMD. We showed that Rab11 did not play a role in viral entry, IRES-mediated protein translation, or viral genome replication, although it co-localized with replication organelles. GTPase-defective Rab11 mutants had no effect on EV-A71 replication, ruling out Rab11 involvement in intracellular trafficking of viral or host components. Instead, reduced VP2:VP0 ratio in siRab11-treated cells supported a role in provirion maturation. Co-immunoprecipitation and mass spectrometry revealed chaperones as Rab11 top interacting partners during infection. Among which, CCT8 subunit of the chaperone complex TRiC/CCT was found to interact with viral structural proteins specifically. Together, this study describes a novel, unconventional role for Rab11 during viral infection, where it participates in the complex process of virus morphogenesis by recruiting essential chaperone proteins.