bluuebunny/crossref_metadata_2025 huggingface dataset:
Clinical profile and immediate outcome of Multisystem Inflammatory Syndrome in Children (MIS-C) associated with Covid-19: a multicentric study
Authors: Sethy Geetanjali , Mishra Bibhudatta , Jain Mukesh Kumar , Patnaik Sibabratta , Mishra Reshmi , Behera Jyoti Ranjan , Sahoo Bandya
Published: June 2021

STRUCTURED ABSTRACT

INTRODUCTION

Following an asymptomatic or mildly symptomatic Corona virus disease (COVID 19), otherwise healthy children, may develop serious manifestations in form of cardiac, neurological, respiratory, gastrointestinal and dermatologic dysfunction. Many such cases were being observed in Odisha, an eastern state of India and reported from different health care facilities. We related these unexplained serious manifestations to Multisystem Inflammatory Syndrome associated with COVID 19 (MIS-C) and planned this study.

METHODS

This retrospective observational study was carried out in three tertiary care centres: Kalinga Institute of Medical Sciences, Bhubaneswar, MKCG Medical college Berhampur and Jagannath Hospital, Bhubaneswar between July to September of year 2020. Study population include all children from 1 month to 15 years admitted to hospital with MIS-C according to WHO Diagnostic Criteria. All the data were analyzed by SPSS.

RESULTS

A total of 21 children were included in our study. Maximum number of cases were male (76.2%), predominate age group was 6-10 yrs (47.6%). Predominate symptoms /signs in our observation were fever, pain abdomen, seizure and hypotension. Most of these cases were positive for SARS CoV antibody (80.95%). Response to immunotherapy was dramatic. Mortality (9%) of our study is higher to 1.8–3% from western literature. None of our patient had coronary abnormality while 2 had mild cardiac dysfunction at discharge comparable to other studies.

CONCLUSION

MIS-C following exposure to COVID 19 infection in children is a clinical syndrome which needs early suspicion and appropriate intervention to prevent mortality.
In vivo delayed clearance of Plasmodium falciparum malaria independent of kelch13 polymorphisms and with escalating malaria in Bangladesh
Authors: Nima Maisha Khair , Sazed Saiful Arefeen , Mukherjee Angana , Hossainey Muhammad Riadul Haque , Phru Ching Swe , Johora Fatema Tuj , Safeukui Innocent , Saha Anjan , Khan Afsana Alamgir , Prue Marma Aung Swi , Ware Russell E. , Narla Mohandas , Calhoun Barbara , Haque Rashidul , Khan Wasif Ali , Alam Mohammad Shafiul , Haldar Kasturi
Published: June 2021

The emergence of resistance to artemisinin drugs threatens global malaria control. Resistance is widely seen in South East Asia (SEA) and Myanmar, but not comprehensively assessed in Bangladesh. This is due to lack of measuring parasite clearance times in response to drug treatment, a gold standard used to track artemisinin resistance (AR), in the Chittagong Hill Tracts (CHT), where >90% of malaria occurs in Bangladesh. Here we report delay in clinical parasite clearance half-lives > 5 h characteristic of AR, in Bandarban, a south-eastern rural, CHT district with escalating malaria and bordering Myanmar. Thirty-one and 68 malaria patients respectively presented in the clinic in 2018 and 2019, and this increase well correlated to the district-level malaria surge and rise in rainfall, humidity and temperature. A total of 27 patients with uncomplicated Plasmodium falciparum malaria mono-infection, after administration of an artemisinin combination therapy (ACT) showed median (range) parasite clearance half-life and time of 5.6 (1.5 – 9.6) and 24 (12–48) hours (h) respectively. The frequency distribution of parasite clearance half-life and time was bimodal, with a slower parasite clearance of 8 h in 20% of the participants. There was however, no detectable parasitemia 72 h after initiating ACT. Half-life clearance of > 5h, respectively seen in 35% and 40% of participants in 2018 and 2019, lacked in correlation to initial parasitemia, blood count parameters or resistance mutations of PfKelch13 (K13, the major parasite marker of AR). Culture adapted strains await assessment of in vitro resistance and new parasite determinants of AR.
Clathrin-independent endocytic retrieval of SV proteins mediated by the clathrin adaptor AP-2 at mammalian central synapses
Authors: López-Hernández Tania , Takenaka Koh-ichiro , Mori Yasunori , Kongpracha Pornparn , Nagamori Shushi , Haucke Volker , Takamori Shigeo
Published: June 2021

Neurotransmission is based on the exocytic fusion of synaptic vesicles (SVs) followed by endocytic membrane retrieval and the reformation of SVs. Conflicting models have been proposed regarding the mechanisms of SV endocytosis, most notably clathrin/ AP-2-mediated endocytosis and clathrin-independent ultrafast endocytosis. Partitioning between these pathways has been suggested to be controlled by temperature and stimulus paradigm. We report on the comprehensive survey of six major SV proteins to show that SV endocytosis in hippocampal neurons at physiological temperature occurs independent of clathrin while the endocytic retrieval of a subset of SV proteins including the vesicular transporters for glutamate and GABA depend on sorting by the clathrin adaptor AP-2. Our findings highlight a clathrin-independent role of the clathrin adaptor AP-2 in the endocytic retrieval of select SV cargos from the presynaptic cell surface and suggest a unified model for the endocytosis of SV membranes at mammalian central synapses.
Phylogenetic network analysis revealed the recombinant origin of the SARS-CoV-2 VOC202012/01 (B.1.1.7) variant first discovered in U.K.
Authors: Xie Xianfa , Lewis Teash-Juan , Green Nikoli , Wang Zhenping
Published: June 2021

The emergence of new variants of the SARS-CoV-2 virus poses serious problems to the control of the current COVID-19 pandemic. Understanding how the variants originate is critical for effective control of the spread of the virus and the global pandemic. The study of the virus evolution so far has been dominated by phylogenetic tree analysis, which however is inappropriate for a few important reasons. Here we used phylogenetic network approach to study the origin of the VOC202012/01 (Alpha) or so-called UK variant (PANGO Lineage B.1.1.7). The multiple network analyses using different methods consistently revealed that the VOC202012/01 variant was a result of recombination, in contrast to the common assumption that the variant evolved from step-wise mutations in a linear order. The study provides an example for the power and application of phylogenetic network analysis in studying virus evolution, which can be applied to study the evolutionary processes leading to the emergence of other variants of the SARS-CoV-2 virus as well as many other viruses.

Significance

The emergence of new variants of the SARS-CoV-2 virus, including the Alpha variant first found in U.K., poses serious challenges to the control of the current COVID-19 pandemic. Understanding how new variant originated is paramount to end the pandemic as effectively and quickly as possible. The dominant phylogenetic tree approach to study virus evolution has been inadequate and even misleading. Here we used a phylogenetic network approach to study the origin of the VOC202012/01 (Alpha) variant which was first reported in U.K. last year but has soon spread into many other countries, leading to dramatic increase in infection and death. Multiple analyses consistently revealed that the variant originated through recombination of pre-existing virus strains, highlighting an important but largely ignored mechanism in the evolution of the SARS-CoV-2 virus so far.
Experience-dependent weakening of callosal synaptic connections in the absence of postsynaptic FMRP
Authors: Zhang Zhe , Gibson Jay R. , Huber Kimberly M.
Published: June 2021

Reduced structural and functional interhemispheric connectivity correlates with the severity of Autism Spectrum Disorder (ASD) behaviors in humans. Little is known of how ASD-risk genes regulate callosal connectivity. Here we show that Fmr1, whose loss-of-function leads to Fragile X Syndrome (FXS), cell autonomously promotes maturation of callosal excitatory synapses between somatosensory barrel cortices in mice. Postnatal, cell-autonomous deletion of Fmr1 in postsynaptic Layer (L) 2/3 or L5 neurons results in a selective weakening of AMPA receptor- (R), but not NMDA receptor-, mediated callosal synaptic function, indicative of immature synapses. Sensory deprivation by contralateral whisker trimming normalizes callosal input strength, suggesting that experience-driven activity of postsynaptic Fmr1 KO L2/3 neurons weakens callosal synapses. In contrast to callosal inputs, synapses originating from local L4 and L2/3 circuits are normal, revealing an input-specific role for postsynaptic Fmr1 in regulation of synaptic connectivity within local and callosal neocortical circuits. These results suggest direct cell autonomous and postnatal roles for FMRP in development of specific cortical circuits and suggest a synaptic basis for long-range functional underconnectivity observed in FXS patients.
Bacterial swarm-mediated phage transportation disrupts a biofilm inherently protected from phage penetration
Authors: Ratheesh Nichith K. , Calderon Cole A. , Zdimal Amanda M. , Shrivastava Abhishek
Published: June 2021

The treatment of chronic bacterial infections by phages has shown promise in combating antimicrobial resistance. A typical phage particle is at least an order of magnitude larger than an antibiotic molecule. Hence, phages diffuse slower than antibiotics, and can also get trapped in the polymeric mesh of biofilm matrix. By tracking fluorescently labeled lambda phages that do not infect Capnocytophaga gingivalis, a bacterium abundant in the human oral microbiota, we demonstrate active phage transportation by a C. gingivalis swarm. As a result, the rate of disruption of the prey of lambda phage i.e., an Escherichia coli colony, increases 10 times. C. gingivalis drills tunnels within a curli fiber containing E. coli biofilm and increase the efficiency of phage penetration. This provides evidence for a novel mechanism of phage-bacterial warfare.
Use of hiPSC-derived cardiomyocytes to rule out proarrhythmic effects of drugs: the case of hydroxychloroquine in COVID-19
Authors: Sala Luca , Leonov Vladislav , Mura Manuela , Moretti Alessandra , Crotti Lia , Gnecchi Massimiliano , Schwartz Peter J
Published: June 2021

In the early phases of the COVID-19 pandemic, drug repurposing was widely used to identify compounds that could improve the prognosis of symptomatic patients infected by SARS-CoV-2. Hydroxychloroquine (HCQ) was one of the first drugs used to treat COVID-19 patients due to its supposed capacity of inhibiting SARS-CoV-2 infection and replication in vitro.

While its efficacy is debated, HCQ has been associated with QT interval prolongation and potentially Torsades de Pointes, especially in patients predisposed to developing drug-induced Long QT Syndrome (LQTS) as silent carriers of variants associated with congenital LQTS. If confirmed, these effects represent a limitation to the at-home use of HCQ for COVID-19 infection as adequate ECG monitoring may be challenging.

We investigated the proarrhythmic profile of HCQ with Multi-Electrode Arrays after subchronic exposure of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from two healthy donors, one asymptomatic and two symptomatic LQTS patients.

We demonstrate that: I) HCQ induced a concentration-dependent Field Potential Duration (FPD) prolongation in vitro and triggered arrhythmias that halted the beating at high concentration. II) hiPSC-CMs from healthy or asymptomatic carriers tolerated higher concentrations of HCQ and showed lower susceptibility to HCQ-induced electrical abnormalities regardless of baseline FPD values. These findings agree with the clinical safety records of HCQ and demonstrated that hiPSC-CMs potentially discriminates symptomatic vs asymptomatic mutation carriers through pharmacological interventions. Disease-specific cohorts of hiPSC-CMs may be a valid preliminary addition to quickly assess drug safety in vulnerable populations, offering rapid preclinical results with valuable translational relevance for precision medicine.
RNF168-mediated localization of BARD1 recruits the BRCA1-PALB2 complex to DNA damage
Authors: Krais John J. , Wang Yifan , Patel Pooja , Basu Jayati , Bernhardy Andrea J. , Johnson Neil
Published: June 2021

DNA damage prompts a diverse range of alterations to the chromatin landscape. The RNF168 E3 ubiquitin ligase catalyzes the mono-ubiquitination of histone H2A at lysine (K)13/15 (mUb-H2A), forming a binding module for DNA repair proteins. BRCA1 promotes homologous recombination (HR), in part, through its interaction with PALB2, and the formation of a larger BRCA1-PALB2-BRCA2-RAD51 (BRCA1-P) complex. The mechanism by which BRCA1-P is recruited to chromatin surrounding DNA breaks is unclear. In this study, we reveal that an RNF168-governed signaling pathway is responsible for localizing the BRCA1-P complex to DNA damage. Using mice harboring a Brca1CC (coiled coil) mutation that blocks the Brca1-Palb2 interaction, we uncovered an epistatic relationship between Rnf168- and Brca1CC alleles, which disrupted development, and reduced the efficiency of Palb2-Rad51 localization. Mechanistically, we show that RNF168-generated mUb-H2A recruits BARD1 through a BRCT domain ubiquitin-dependent recruitment motif (BUDR). Subsequently, BARD1-BRCA1 accumulate PALB2-RAD51 at DNA breaks via the CC domain-mediated BRCA1-PALB2 interaction. Together, these findings establish a series of molecular interactions that connect the DNA damage signaling and HR repair machinery.
Comparing Imaging Biomarkers of Cerebral Edema after TBI in Young Adult Male and Female Rats
Authors: Minchew Heather M , Ferren Sadie L , Christian Sarah K , Hu Jinxiang , Keselman Paul , Brooks William M , Andrews Brian T , Harris Janna L
Published: June 2021

Traumatic brain injury (TBI) is one of the leading causes of death and disability worldwide. Cerebral edema following TBI is known to play a critical role in injury severity and prognosis. In the current study we used multimodal magnetic resonance imaging (MRI) to assess cerebral edema 24 hours after unilateral contusive TBI in male and female rats. We then directly quantified brain water content in the same subjects ex vivo. We found that both males and females had similarly elevated T2 values after TBI compared with sham controls. Apparent diffusion coefficient (ADC) was more variable than T2 and did not show significant injury effects in males or females. Brain water was elevated in male TBI rats compared with sham controls, but there was no difference between female TBI and sham groups. Notably, MRI biomarkers of edema were more closely correlated with brain water in male rats; female rats did not show any relationship between brain water and T2 or ADC. These observations raise questions about the interpretation of radiological findings traditionally interpreted as edema in female TBI patients. A better understanding of sex differences and similarities in the pathophysiology of post-traumatic edema is needed to help improve patient management and the development of effective treatment strategies for men and women.
Functional diversification gave rise to allelic specialization in a rice NLR immune receptor pair
Authors: De la Concepcion Juan Carlos , Benjumea Javier Vega , Białas Aleksandra , Terauchi Ryohei , Kamoun Sophien , Banfield Mark J.
Published: June 2021

Cooperation between receptors from the NLR superfamily is important for intracellular activation of immune responses. NLRs can function in pairs that, upon pathogen recognition, trigger hypersensitive cell death and stop pathogen invasion. Natural selection drives specialization of host immune receptors towards an optimal response, whilst keeping a tight regulation of immunity in the absence of pathogens. However, the molecular basis of co-adaptation and specialization between paired NLRs remains largely unknown. Here, we describe functional specialization in alleles of the rice NLR pair Pik that confers resistance to strains of the blast fungusMagnaporthe oryzaeharbouring AVR-Pik effectors. We revealed that matching pairs of allelic Pik NLRs mount effective immune responses whereas mismatched pairs lead to autoimmune phenotypes, a hallmark of hybrid necrosis in both natural and domesticated plant populations. We further showed that allelic specialization is largely underpinned by a single amino acid polymorphism that determines preferential association between matching pairs of Pik NLRs. These results provide a framework for how functionally linked immune receptors undergo co-adaptation to provide an effective and regulated immune response against pathogens. Understanding the molecular constraints that shape paired NLR evolution has implications beyond plant immunity given that hybrid necrosis can drive reproductive isolation.
ROCK1 regulates insulin secretion from β-cells
Authors: Sung Byung-Jun , Lim Sung-Bin , Kim Jae Hyeon , Yang Won Mo , Kulkarni Rohit N. , Kim Young-Bum , Lee Moon-Kyu
Published: June 2021

Objective

The endocrine pancreatic β-cells play a pivotal role in the maintenance of whole-body glucose homeostasis and its dysregulation is a consistent feature in all forms of diabetes. However, knowledge of intracellular regulators that modulate β-cell function remains incomplete. We investigated the physiological role of ROCK1 in the regulation of insulin secretion and glucose homoeostasis.

Methods

Mice lacking ROCK1 in pancreatic β-cells (RIP-Cre; ROCK1loxP/loxP, β-ROCK1-/-) were studied. Glucose and insulin tolerance tests as well as glucose-stimulated insulin secretion (GSIS) were measured. Insulin secretion response to a direct glucose or pyruvate or pyruvate kinase (PK) activator stimulation in isolated islets from β-ROCK1-/-mice or β-cell lines with knockdown of ROCK1 were also evaluated. Proximity ligation assay was performed to determine the physical interactions between PK and ROCK1.

Results

Mice with a deficiency of ROCK1 in pancreatic β-cells exhibited significantly increased blood glucose levels and reduced serum insulin without changes in body weight. Interestingly, β-ROCK1-/-mice displayed progressive impairment of glucose tolerance while maintaining insulin sensitivity mostly due to impaired GSIS. Consistently, GSIS was markedly decreased in ROCK1-deficient islets and ROCK1 knockdown INS-1 cells. Concurrently, ROCK1 blockade led to a significant decrease in intracellular calcium levels, ATP levels, and oxygen consumption rates in isolated islets and INS-1 cells. Treatment of ROCK1-deficient islets or ROCK1 knockdown β-cells either with pyruvate or a PK activator rescued the impaired GSIS. Mechanistically, we observed that ROCK1 binding to PK is greatly enhanced by glucose stimulation in β-cells.

Conclusions

Our findings demonstrate that β-cell ROCK1 is essential for glucose-stimulated insulin secretion and maintenance of glucose homeostasis and that ROCK1 acts as an upstream regulator of glycolytic pyruvate kinase signaling.
CaProDH2-mediated modulation of proline metabolism confers tolerance to Ascochyta in chickpea under drought
Authors: Patil Mahesh , Pandey Prachi , Irrulappan Vadivelmurugan , Singh Anuradha , Verma Praveen , Ranjan Ashish , Senthil-Kumar Muthappa
Published: June 2021

Drought and leaf blight caused by the fungus Ascochyta rabiei often co-occur in chickpea (Cicer arietinum)-producing areas. While the responses of chickpea to either drought or A. rabiei infection have been extensively studied, their combined effect on plant defense mechanisms is unknown. Fine modulation of stress-induced signaling pathways under combined stress is an important stress adaptation mechanism that warrants a better understanding. Here we show that drought facilitates resistance against A. rabiei infection in chickpea. The analysis of proline levels and gene expression profiling of its biosynthetic pathway under combined drought and A. rabiei infection revealed the gene encoding proline dehydrogenase (CaProDH2) as a strong candidate conferring resistance to A. rabiei infection. Transcript levels of CaProDH2, pyrroline-5-carboxylate (P5C) quantification, and measurement of mitochondrial reactive oxygen species (ROS) production showed that fine modulation of the proline–P5C cycle determines the observed resistance. In addition, CaProDH2-silenced plants lost basal resistance to A. rabiei infection induced by drought, while overexpression of the gene conferred higher resistance to the fungus. We suggest that the drought-induced accumulation of proline in the cytosol helps maintain cell turgor and raises mitochondrial P5C contents by a CaProDH2-mediated step, which results in ROS production that boosts plant defense responses and confers resistance to A. rabiei infection. Our findings indicate that manipulating the proline–P5C pathway may be a possible strategy for improving stress tolerance in plants suffering from combined drought and A. rabiei infection.
Multiplexed single-molecule analysis of human telomerase synthesizing DNA
Authors: Hentschel Jendrik , Choi Junhong , Bagshaw Clive R. , Lapointe Christopher P. , Wang Jinfan , Jansson Linnea I. , Badstuebner Mareike , Puglisi Joseph D. , Stone Michael D.
Published: June 2021

Genomic stability in proliferating cells critically depends on telomere maintenance by telomerase reverse transcriptase. Here we developed a real-time single-molecule RNA sequencing approach that visualizes telomerase catalysis and structural dynamics at single-nucleotide resolution using FRET and zero-mode waveguides. The method permits direct detection of dynamic steps and structural states throughout the telomerase catalytic cycle and can be generalized to other nucleic acid polymerase systems.
Deep parallel characterization of AAV tropism and AAV-mediated transcriptional changes via single-cell RNA sequencing
Authors: Brown David , Altermatt Michael , Dobreva Tatyana , Chen Sisi , Wang Alexander , Thomson Matt , Gradinaru Viviana
Published: June 2021

Engineered variants of recombinant adeno-associated viruses (rAAVs) are being developed rapidly to meet the need for gene-therapy delivery vehicles with particular cell-type and tissue tropisms. While high-throughput AAV engineering and selection methods have generated numerous variants, subsequent tropism and response characterization have remained low throughput and lack resolution across the many relevant cell and tissue types. To fully leverage the output of these large screening paradigms across multiple targets, we have developed an experimental and computational single-cell RNA sequencing (scRNA-seq) pipeline for in vivo characterization of barcoded rAAV pools at unprecedented resolution. Using our platform, we have corroborated previously reported viral tropisms and discovered unidentified AAV capsid targeting biases. As expected, we observed that the tropism profile of AAV.CAP-B10 in mice was shifted toward neurons and away from astrocytes when compared with AAV-PHP.eB. Our transcriptomic analysis revealed that this neuronal bias is mainly due to increased targeting efficiency for glutamatergic neurons, which we confirmed by RNA fluorescence in situ hybridization. We further uncovered cell subtype tropisms of AAV variants in vascular and glial cells, such as low transduction of pericytes and Myoc+ astrocytes. Additionally, we have observed cell-type-specific responses to systemic AAV-PHP.eB administration, such as upregulation of genes involved in p53 signaling in endothelial cells three days post-injection, which return to control levels by day twenty-five. Such ability to parallelize the characterization of AAV tropism and simultaneously measure the transcriptional response of transduction will facilitate the advancement of safe and precise gene delivery vehicles.
AP-4 regulates neuronal lysosome composition, function, and transport via regulating export of critical lysosome receptor proteins at the trans-Golgi network
Authors: Majumder Piyali , Edmison Daisy , Rodger Catherine , Patel Sruchi , Reid Evan , Gowrishankar Swetha
Published: June 2021

The adaptor protein complex-4 or AP-4 is known to mediate autophagosome maturation through regulating sorting of transmembrane cargo such as ATG9A at the Golgi. There is a need to understand AP-4 function in neurons, as mutations in any of its four subunits cause a complex form of hereditary spastic paraplegia (HSP) with intellectual disability. While AP-4 has been implicated in regulating trafficking and distribution of cargo such as ATG9A and APP, little is known about its effect on neuronal lysosomal protein traffic, lysosome biogenesis and function. In this study, we demonstrate that in human iPSC-derived neurons AP-4 regulates lysosome composition, function and transport via regulating export of critical lysosomal receptors, including Sortilin 1, from the trans-Golgi network to endo-lysosomes. Additionally, loss of AP-4 causes endo-lysosomes to stall and build up in axonal swellings potentially through reduced recruitment of retrograde transport machinery to the organelle. These findings of axonal lysosome build-up are highly reminiscent of those observed in Alzheimer’s disease as well as in neurons modelling the most common form of HSP, caused by spastin mutations. Our findings implicate AP-4 as a critical regulator of neuronal lysosome biogenesis and altered lysosome function and axonal endo-lysosome transport as an underlying defect in AP-4 deficient HSP.
Cytoplasmic Colocalization of Granulins and TDP-43 Prion-like Domain Involves Electrostatically Driven Coacervation Tuned by the Redox State of Cysteines
Authors: Bhopatkar Anukool A. , Dhakal Shailendra , Rangachari Vijayaraghavan
Published: June 2021

Cytoplasmic inclusions containing aberrant proteolytic fragments of TDP-43 are associated with frontotemporal lobar degeneration (FTLD) and other related pathologies. In FTLD, TDP-43 is translocated into the cytoplasm and proteolytically cleaved to generate a prion-like domain (PrLD) containing C-terminal fragments (C25 and C35) that form toxic inclusions. Under stress, TDP-43 partitions into membraneless organelles called stress granules (SGs) by coacervating with RNA and other proteins. We were interested in understanding if and how cysteine-rich granulins (GRNs 1-7), which are the proteolytic products of a genetic risk factor in FTLD called progranulin, interact with TDP-43. We show that extracellular GRNs internalize and colocalize with PrLD as puncta in the cytoplasm of neuroblastoma cells but show no presence in SGs. In addition, we show GRNs and PrLD coacervate to undergo liquid-liquid phase separation (LLPS) or form gel- or solid-like aggregates. Identification of the sequence determinants within GRNs for the observed phase transitions reveal the negative charges to be the drivers of LLPS modulated by the positive charges and the redox state of cysteines. Furthermore, RNA and GRNs compete and expunge one another from PrLD condensates, providing a basis for GRN’s absence in SGs. Together, the results illustrate the potential mechanisms by which extracellular GRNs, formed during chronic inflammatory conditions, could internalize, and modulate cytoplasmic TDP-43 inclusions in proteinopathies.
A steroid hormone regulates growth in response to oxygen availability
Authors: Kapali George P. , Callier Viviane , Broeker Hailey , Tank Parth , Gascoigne Samuel J.L. , Harrison Jon F , Shingleton Alexander W.
Published: June 2021

In almost all animals, physiologically low oxygen (hypoxia) during development slows growth and reduces adult body size1–3. The developmental mechanisms that determine growth under hypoxic conditions are, however, poorly understood. One hypothesis is that the effect of hypoxia on growth and final body size is a non-adaptive consequence of the cell-autonomous effects of hypoxia on cellular metabolism. Alternatively, the effect may be an adaptive coordinated response mediated through systemic physiological mechanisms. Here we show that the growth and body size response to moderate hypoxia (10% O2) in Drosophila melanogaster is systemically regulated via the steroid hormone ecdysone, acting partially through the insulin-binding protein Imp-L2. Ecdysone is necessary to reduce growth in response to hypoxia: hypoxic growth suppression is ameliorated when ecdysone synthesis is inhibited. This hypoxia-suppression of growth is mediated by the insulin/IGF-signaling (IIS) pathway. Hypoxia reduces systemic IIS activity and the hypoxic growth-response is eliminated in larvae with suppressed IIS. Further, loss of Imp-L2, an ecdysone-response gene that suppresses systemic IIS, significantly reduces the negative effect of hypoxia on final body size. Collectively, these data indicate that growth suppression in hypoxic Drosophila larvae is accomplished by systemic endocrine mechanisms rather than direct suppression of tissue aerobic metabolism.
Mitogen Kinase Kinase (MKK7) controls cytokine production in vitro and in vivo in mice
Authors: Caliz Amada D. , Yoo Hyung-Jin , Vertii Anastassiia , Tournier Cathy , Davis Roger J. , Keaney John F. , Kant Shashi
Published: June 2021

Mitogen kinase kinase 4 (MKK4) and Mitogen kinase kinase 7 (MKK7) are members of the MAP2K family which can activate downstream mitogen-activated protein kinases (MAPKs). MKK4 has been implicated in the activation of both, c-Jun N-terminal Kinase (JNK) and p38 MAPK, whereas MKK7 only activates JNK in response to different stimuli. The stimuli as well as cell type determine the choice of MAP2K member that mediates the response. In a variety of cell types, the MKK7 contributes to the activation of downstream MAPKs, JNK, which is known to regulate essential cellular processes, such as cell death, differentiation, stress response, and cytokine secretion. Previous studies have implicated the role of MKK7 in stress signaling pathways and cytokine production. However, little is known about the degree to which MKK7 and MKK4 contributes to innate immune response in macrophages as well as during inflammation in vivo. To address this question and elucidate the role of MKK7 and MKK4 in macrophage and in vivo, we developed MKK7- and MKK4-deficient mouse models with tamoxifen-inducible Rosa26 CreERT. This study reports that MKK7 is required for JNK activation both in vitro and in vivo. Additionally, we demonstrated that MKK7 in macrophages is necessary for LPS induced cytokine production and migration which appears to be a major contributor to the inflammatory response in vivo. Whereas MKK4 plays a significant but minor role in cytokine production in vivo.
MassGenie: a transformer-based deep learning method for identifying small molecules from their mass spectra
Authors: Shrivastava Aditya Divyakant , Swainston Neil , Samanta Soumitra , Roberts Ivayla , Muelas Marina Wright , Kell Douglas B.
Published: June 2021

The ‘inverse problem’ of mass spectrometric molecular identification (‘given a mass spectrum, calculate the molecule whence it came’) is largely unsolved, and is especially acute in metabolomics where many small molecules remain unidentified. This is largely because the number of experimentally available electrospray mass spectra of small molecules is quite limited. However, the forward problem (‘calculate a small molecule’s likely fragmentation and hence at least some of its mass spectrum from its structure alone’) is much more tractable, because the strengths of different chemical bonds are roughly known. This kind of molecular identification problem may be cast as a language translation problem in which the source language is a list of high-resolution mass spectral peaks and the ‘translation’ a representation (for instance in SMILES) of the molecule. It is thus suitable for attack using the deep neural networks known as transformers. We here present MassGenie, a method that uses a transformer-based deep neural network, trained on ~6 million chemical structures with augmented SMILES encoding and their paired molecular fragments as generatedin silico, explicitly including the protonated molecular ion. This architecture (containing some 400 million elements) is used to predict the structure of a molecule from the various fragments that may be expected to be observed when some of its bonds are broken. Despite being given essentially no detailed nor explicit rules about molecular fragmentation methods, isotope patterns, rearrangements, neutral losses, and the like, MassGenie learns the effective properties of the mass spectral fragment and valency space, and can generate candidate molecular structures that are very close or identical to those of the ‘true’ molecules. We also use VAE-Sim, a previously published variational autoencoder, to generate candidate molecules that are ‘similar’ to the top hit. In addition to using the ‘top hits’ directly, we can produce a rank order of these by ‘round-tripping’ candidate molecules and comparing them with the true molecules, where known. As a proof of principle, we confine ourselves to positive electrospray mass spectra from molecules with a molecular mass of 500Da or lower. The transformer method, applied here for the first time to mass spectral interpretation, works extremely effectively both for mass spectra generatedin silicoand on experimentally obtained mass spectra from pure compounds. The ability to create and to ‘learn’ millions of fragmentation patternsin silico, and therefrom generate candidate structures (that do not have to be in existing libraries)directly, thus opens up entirely the field ofde novosmall molecule structure prediction from experimental mass spectra.
Xrn1 is a deNADding Enzyme Modulating Mitochondrial NAD Levels
Authors: Sharma Sunny , Yang Jun , Grudzien-Nogalska Ewa , Kiledjian Megerditch
Published: June 2021

The existence of non-canonical nicotinamide adenine diphosphate (NAD) 5’-end capped RNAs is now well established. Nevertheless, the biological function of this nucleotide metabolite cap remains elusive. Here, we show that the yeast Saccharomyces cerevisiae cytoplasmic 5’-end exoribonuclease Xrn1 is also a NAD cap decapping (deNADding) enzyme that releases intact NAD and subsequently degrades the RNA. The significance of Xrn1 deNADding is evident in a deNADding deficient Xrn1 mutant that still retains its 5’-monophosphate exonuclease activity. This mutant reveals Xrn1 deNADding is necessary for normal growth on non-fermenting sugar and is involved in modulating mitochondrial NAD-capped RNA levels and in turn intramitochondrial NAD levels. Our findings uncover a functional role for mitochondrial NAD-capped RNAs as a reservoir to maintain overall NAD homeostasis. We propose NAD-capped RNAs function as a cistern for mitochondrial NAD with Xrn1 serving as a rheostat for NAD-capped RNAs.
Inositolphosphate glycans and a fucosylated xyloglucan oligosaccharide are accumulated upon Arabidopsis thaliana/ Botrytis cinerea infection
Authors: Voxeur Aline , Sechet Julien , Vernhettes Samantha
Published: June 2021

In mammals, insulin is involved in controlling blood glucose levels and its role in modulating immunity is being more and more documented. This hormone promotes the release of inositolphosphate glycans (IPG) which act as mediators. In plants, one IG has already been identified in plant culture cells (Smith and Fry, 1999; Smith et al., 1999) but, to our knowledge, no IPG have been yet identified. Here, we discovered 7 IPG that are accumulated upon Arabidopsis thaliana-Botrytis cinerea interaction, concomitantly with oligogalacturonides and a fucosylated xyloglucan oligosaccharide. Further structural characterization showed that they come from the hydrolysis of polar heads of Serie A to H glycosylinositol phosphorylceramides presumably via a phospholipase C activity. Taken together with the emerging role of insulin as immune regulator, these results question the role of IPG as damage associated molecular pattern both in animal and plant kingdoms.
A metabolically stable PET tracer for imaging synaptic vesicle protein 2A: Synthesis and preclinical characterization of [18F]SDM-16
Authors: Zheng Chao , Holden Daniel , Zheng Ming-Qiang , Pracitto Richard , Wilcox Kyle C. , Lindemann Marcel , Felchner Zachary , Zhang Li , Tong Jie , Fowles Krista , Finnema Sjoerd J. , Nabulsi Nabeel , Carson Richard E. , Huang Yiyun , Cai Zhengxin
Published: June 2021

Purpose

To investigate the synaptic vesicle glycoprotein 2A (SV2A) expression in the whole central nervous system and peripheral tissues, a metabolically stable SV2A radiotracer is desirable to minimize a potential confounding effect of radiometabolites. The aim of this study was to develop and evaluate a metabolically stable SV2A radiotracer, [18F]SDM-16, in nonhuman primate brains.

Methods

The racemic SDM-16 (4-(3,5-difluorophenyl)-1-((2-methyl-1H-imidazol-1-yl)methyl)pyrrolidin-2-one) was synthesized and assayed for in vitro SV2A binding affinity. We synthesized the enantiopure [18F]SDM-16 using the corresponding arylstannane precursor. Nonhuman primate brain PET was performed on a FOCUS 220 system. Arterial blood was drawn for metabolite analysis and construction of plasma input function. Regional time-activity curves (TACs) were evaluated with the one-tissue compartment (1TC) model to obtain the volume of distribution (VT). Binding potential (BPND) was calculated using either the nondisplaceable volume of distribution (VND) or the centrum semiovale (CS) as the reference region.

Results

Racemic SDM-16 was synthesized in 3 steps with 44% overall yield and has high affinity (Ki = 3.7 nM) to human SV2A. [18F]SDM-16 was prepared in greater than 99% radiochemical and enantiomeric purity. This radiotracer displayed high specific binding in brain and was metabolically more stable than other SV2A PET tracers. The plasma free fraction (fP) of [18F]SDM-16 was 69%, which was higher than those of [11C]UCB-J (46%), [18F]SynVesT-1 (43%), [18F]SynVesT-2 (41%), and [18F]UCB-H (43%). The TACs were well described with the 1TC. The averaged test-retest variability (TRV) was −9±8%, and averaged absolute TRV (aTRV) was 10±7% for all analyzed brain regions.

Conclusion

We have successfully synthesized a metabolically stable and high affinity SV2A PET tracer, [18F]SDM-16, which showed high specific and reversible binding in the NHP brain. [18F]SDM-16 may have potential application in the visualization and quantification of SV2A beyond the brain.
Efficient tagging and purification of endogenous proteins for structural studies by single particle cryo-EM
Authors: Zhao Jianhua , Makhija Suraj , Huang Bo , Cheng Yifan
Published: June 2021

A major bottleneck in structural biology is producing biologically relevant samples at sufficient quantities. This is particularly true for large protein assemblies where conventional techniques of gene overexpression require substantial optimization, hampering structural studies and drug development efforts. Here we describe a method combining CRISPR/Cas gene editing and fluorescence cell sorting to rapidly tag and purify endogenous human proteins from cell lines, enabling structural analysis of native proteins that are properly folded and assembled. We applied this approach to study the human proteasome from HEK cells and rapidly determined structures of major proteasomal complexes. Structures of the PA28-20S complex reveal the native subunit stoichiometry of PA28 and a distinct functional state of the complex. The efficient strategy for tagging and extracting endogenous proteins described here will enable the structural study of many challenging targets and provide more biologically relevant samples for research and therapeutic development.
FRET and LRET Biosensors for Cell-based Imaging and Screening of Rac1 Activation
Authors: Pham Ha , Soflaee Mona Hoseini , Karginov Andrei V , Miller Lawrence W.
Published: June 2021

Rac1 is a key regulator of several cell signaling pathways and dysregulated Rac1 activation has been implicated in cancer. Genetically encoded Förster resonance energy transfer (FRET) biosensors with enhanced dynamic range enabled live cell fluorescence imaging of Rac1 activity and a cell lysate-based assay of Rac1 inhibition in 96-well plates. We prepared HEK293 cell lines that stably expressed polypeptides with a general domain sequence (N- to C-terminus) of 1) FRET acceptor; 2) Rac/Cdc42 binding domain of human p21 protein kinase A (residues 68-150); 3) a linker domain; 4) FRET donor; and 5) full-length Rac1. Activated Rac1 binds to the protein kinase A domain, bringing donors and acceptors close together to increase FRET. We evaluated the effects on FRET signal dynamic range of alpha helical linkers comprised of alternating repeats of roughly four glutamate and four arginine or lysine residues. So-called ER/K linkers had limited effects on conventional FRET biosensors that incorporated the fluorescent protein (FP) pairs mCerulean/YPet, or mTFP1(cp227)/mVenus(cp229). Fluorometric measurements of cells that co-expresssed biosensors with positive (TIAM1) or negative (RhoGDI) Rac1 regulators revealed significant dynamic range enhancement in only one FP construct (mCerulean/YPet with 20 nm ER/K linker) relative to an analogous structure that incorporated an unstructured linker. We transfected this construct into a cell line that stably expressed a rapamycin-inducible c-Src analog (RapR-Src) and observed activation of Rac1 at protruding edges following rapamycin stimulation. In cells that expressed lanthanide-based FRET (LRET biosensors) that incorporated a luminescent terbium complex donor and GFP fluorescent acceptor, time-gated luminescence (TGL) measurements showed substantial gains in dynamic range that increased with linker length (up to 1200%). We robustly detected small molecule Rac1 inhibition following lysis of LRET biosensor-expressing cells grown directly in 96-well plates. The results herein highlight the potential of FRET and LRET biosensors with ER/K linkers for cell-based imaging and screening of protein activities.
Testing methods for quantifying Monte Carlo variation for categorical variables in Probabilistic Genotyping
Authors: Bright Jo-Anne , Taylor Duncan , Curran James , Buckleton John
Published: June 2021

Two methods for applying a lower bound to the variation induced by the Monte Carlo effect are trialled. One of these is implemented in the widely used probabilistic genotyping system, STRmix™. Neither approach is giving the desired 99% coverage. In some cases the coverage is much lower than the desired 99%. The discrepancy (i.e. the distance between theLRcorresponding to the desired coverage and theLRobserved coverage at 99%) is not large. For example, the discrepancy of 0.23 for approach 1 suggests the lower bounds should be moved downwards by a factor of 1.7 to achieve the desired 99% coverage.

Although less effective than desired these methods provide a layer of conservatism that is additional to the other layers. These other layers are from factors such as the conservatism within the sub-population model, the choice of conservative measures of co-ancestry, the consideration of relatives within the population and the resampling method used for allele probabilities, all of which tend to understate the strength of the findings.

Highlights

Two methods for quantifying Monte Carlo variability are tested,

Both give less than the desired 99% coverage,

The magnitude of possible discrepancy is small,

For example anLRof 4.3 × 1011could be reported as 1.8 × 1012

AnLRof 18 could be reported as 22.
Environmental RNAi-based reverse genetics in the predatory mite Neoseiulus californicus: towards improved methods of biological control
Authors: Ghazy Noureldin Abuelfadl , Suzuki Takeshi
Published: June 2021

The predatory mite Neoseiulus californicus (McGregor) (Mesostigmata: Phytoseiidae) has been commercialized by manufacturers in the pest control industry and is used worldwide as a natural enemy of spider mites. However, because its genome has not been sequenced, reverse genetics techniques that could be used to analyze gene function have not been established. Here we partially sequenced the gene that encodes the vacuolar-type H+-ATPase (V-ATPase), an ATP-dependent proton pump, in N. californicus (NcVATPase) and then conducted a functional analysis using environmental RNA interference (eRNAi) by orally administering sequence-specific exogenous dsRNA (dsRNA-NcVATPase) to larvae and adult females. The larvae treated with dsRNA-NcVATPase took longer to develop and had lower survivorship, fecundity, and offspring viability at the adult stage than those treated with a control dsRNA. Adult females treated with dsRNA-NcVATPase showed significant reductions in survival, fecundity, and prey consumption, and their endogenous gene expression level of NcVATPase was reduced by approximately 65% compared with the control. Our findings suggest that the NcVATPase gene, silencing of which inhibits feeding and reproduction, is an excellent biomarker for investigating the eRNAi mechanism in N. californicus. The highly efficient experimental system of eRNAi established in this study paves the way for applied research using eRNAi to enhance the predatory ability of N. californicus.

Key message

Environmental RNAi-inducing double-stranded RNAs have the potential to improve biological control as well as biopesticide applications.

We investigated the efficacy of eRNAi against the predatory mite Neoseiulus californicus, a major natural enemy of spider mites.

Oral administration of dsRNA targeting NcVATPase decreased the gene expression level, developmental time, survival, fecundity, and prey consumption.

Neoseiulus californicus, which was found to have the high eRNAi effects, can be used as a model for the study on eRNAi-mediated improvement of biological control.
Large structural variations in the haplotype-resolved African cassava genome
Authors: Mansfeld Ben N. , Boyher Adam , Berry Jeffrey C. , Wilson Mark , Ou Shujun , Polydore Seth , Michael Todd P. , Fahlgren Noah , Bart Rebecca S.
Published: June 2021

Cassava (Manihot esculentaCrantz, 2n=36) is a global food security crop. Cassava has a highly heterozygous genome, high genetic load, and genotype-dependent asynchronous flowering. It is typically propagated by stem cuttings and any genetic variation between haplotypes, including large structural variations, is preserved by such clonal propagation. Traditional genome assembly approaches generate a collapsed haplotype representation of the genome. In highly heterozygous plants, this results in artifacts and an oversimplification of heterozygous regions. We used a combination of Pacific Biosciences (PacBio), Illumina, and Hi-C to resolve each haplotype of the genome of a farmer-preferred cassava line, TME7 (Oko-iyawo). PacBio reads were assembled using the FALCON suite. Phase switch errors were corrected using FALCON-Phase and Hi-C read data. The ultra-long-range information from Hi-C sequencing was also used for scaffolding. Comparison of the two phases revealed more than 5,000 large haplotype-specific structural variants affecting over 8 Mb, including insertions and deletions spanning thousands of base pairs. The potential of these variants to affect allele specific expression was further explored. RNA-seq data from 11 different tissue types were mapped against the scaffolded haploid assembly and gene expression data are incorporated into our existing easy-to-use web-based interface to facilitate use by the broader plant science community. These two assemblies provide an excellent means to study the effects of heterozygosity, haplotype-specific structural variation, gene hemizygosity, and allele specific gene expression contributing to important agricultural traits and further our understanding of the genetics and domestication of cassava.

Significance statement

The cassava varieties grown by subsistence farmers in Africa largely differ from the inbred reference genome due to their highly heterozygous nature. We used multiple sequencing technologies to assemble and resolve both haplotypes in TME7, a farmer-preferred cassava line, enabling us to study the considerable haplotypic structural variation in this line.