bluuebunny/crossref_metadata_2025 huggingface dataset:
Synthesis of tin(IV) nitride with spinel structure,
γ
-Sn
3
N
4
, from the elements and its Raman-spectroscopic examination at high pressures
Authors: Zerr Andreas , Miehe Gerhard
Published: August 2023

We report on the synthesis of tin(IV) nitride with spinel structure,
γ
-Sn
3
N
4
, from the elements at high pressures and temperatures using a laser-heated diamond anvil cell, and on the Rietveld refinement of the product structure. The procedure described here is, in our opinion, the most reliable method of obtaining high-purity nitrides which are thermodynamically stable only at high pressures. Raman spectroscopy and powder X-ray diffraction were used to characterize the synthesis products. Pressure dependences of the Raman-band frequencies of
γ
-Sn
3
N
4
were measured and used to determine its average mode Grüneisen parameter, 〈
γ
〉 = 0.95. Using this value, we estimated the thermal-shock resistance of
γ
-Sn
3
N
4
to be about half that of
γ
-Si
3
N
4
, which, in turn, is moderately surpassed by
β
-Si
3
N
4
, known to be highly thermal-shock resistant.

This article is part of the theme issue ‘Exploring the length scales, timescales and chemistry of challenging materials (Part 1)’.
Clustering of fluoride and phosphate ions in bioactive glass from computer simulation
Authors: Christie Jamieson K.
Published: August 2023

In order to understand the nature of ionic clustering in bioactive glass compositions, computer simulation was used to model four different compositions of bioactive glass with various amounts of flouride and phosphate. Fluoride ions were chemically bonded only to sodium and calcium, creating regions rich in fluoride and modifiers, and fluoride clustering was seen to be present in all compositions. The majority of phosphate groups are present as orthophosphate and phosphate clustering is also seen, and shown to be stronger in compositions with a lower phosphate content.

This article is part of the theme issue ‘Exploring the length scales, timescales and chemistry of challenging materials (Part 1)’.
Iron coordination in liquid FeAl
2
O
4
Authors: Drewitt James W. E. , Barnes Adrian C. , Jahn Sandro , Brooker Richard A. , Hennet Louis , Neuville Daniel R. , Fischer Henry E.
Published: August 2023

The structure of aerodynamically levitated liquid

FeAl

2

O

4

was measured by neutron diffraction with isotope substitution (NDIS). Classical and
ab initio
molecular dynamics simulations were performed and their results were found to be in close agreement with each other and the NDIS data. The results reveal that molten

FeAl

2

O

4

may be considered as an ionic liquid without any preference for particular short-range structural motifs.

This article is part of the theme issue ‘Exploring the length scales, timescales and chemistry of challenging materials (Part 1)’.
Effect of water on the glass transition of a potassium-magnesium carbonate melt
Authors: Weidendorfer Daniel , Hess Kai-Uwe , Ruhekenya Ruben M. , Schawe Jürgen E. K. , Wilding Martin C. , Dingwell Donald B.
Published: August 2023

Calorimetric measurements of the glass transition temperatures (Tg) of hydrous carbonate melts are reported on a near-eutectic composition of 55 mol% K2CO3– 45 mol% MgCO3with up to 42 mol% bulk H2O dissolved in the carbonate melt. Hydrous melts were quenched from 750°C to transparent and crystal-free glasses and were subsequently analysed for water content before and after measuringTgby high-sensitivity differential scanning calorimetry. The glass transition and limited fictive temperatures as a function of the water content were determined at 10 K/min cooling/heating rates resulting inTgranging from 245°C at nominally anhydrous conditions to 83°C in the presence of 42 mol% H2O in the glass. Through a generalized Gordon–Taylor analysis, the factorsk(7.27),k0(3.2) and the interaction parameterAx(0.49) were derived. The limited fictive temperature of a hypothetically, zero water containing 55 mol% K2CO3– 45 mol% MgCO3glass is 232 ± 5°C (505 K). The high value of the interaction parameterAindicates strong specific molecular interactions between water and the carbonates in the glassy state and a large decrease in the excess enthalpy of mixing during the conversion of the glassy into the liquid state at the glass transition.

This article is part of the theme issue 'Exploring the length scales, timescales and chemistry of challenging materials (Part 1)'.
Elastic moduli and refractive index of γ-Ge
3
N
4
Authors: Li Chen-Hui , Djemia Philippe , Chigarev Nikolay , Sodki Siham , Roussigné Yves , Manthilake Geeth , Tessier Franck , Raetz Samuel , Gusev Vitalyi E. , Zerr Andreas
Published: August 2023

Germanium nitride, having cubic spinel structure, γ-Ge
3
N
4
, is a wide band-gap semiconductor with a large exciton binding energy that exhibits high hardness, elastic moduli and elevated thermal stability up to approximately 700°C. Experimental data on its bulk and shear moduli (
B
0
and
G
0
, respectively) are strongly limited, inconsistent and, thus, require verification. Moreover, earlier first-principles density functional calculations provided significantly scattering
B
0
values but consistently predicted
G
0
much higher than the so far available experimental value. Here, we examined the elasticity of polycrystalline γ-Ge
3
N
4
, densified applying high pressures and temperatures, using the techniques of laser ultrasonics (LU) and Brillouin light scattering (BLS) and compared with our extended first-principles calculations. From the LU measurements, we obtained its longitudinal- and Rayleigh wave sound velocities and, taking into account the sample porosity, derived
B
0
 = 322(44) GPa and
G
0
 = 188(7) GPa for the dense polycrystalline γ-Ge
3
N
4
. While our calculations underestimated
B
0
by approximately 17%, most of the predicted
G
0
matched well with our experimental value. Combining the LU- and BLS data and taking into account the elastic anisotropy, we determined the refractive index of γ-Ge
3
N
4
in the visible range of light to be
n
 = 2.4, similarly high as that of diamond or GaN, and matching our calculated value.

This article is part of the theme issue 'Exploring the length scales, timescales and chemistry of challenging materials (Part 1)'.
Water and foodborne bacterial pathogens isolated from hospitalized children are extensively resistance against commonly used Penicillin-type antibiotics in developing countries
Authors: Awuor Silas Onyango , Omwenga Eric O. , Mariita Richard M. , Musila Jonathan M. , Mbeke Alexander , Musyoka Thommas M.
Published: August 2023

Diarrheal diseases pose a significant threat to children under five years, ranking as the second leading cause of death in this age group. This is partly attributed to the rise of multidrug-resistant enteric bacteria that has further exacerbated the current global health crisis. We herein report a study aimed at identifying common diarrhea-causing bacteria and assessing their antibiotic susceptibility patterns in children under five seeking medical care at Muhoroni County Hospital. Between January and June 2016, a total of 196 under-five children seeking medical care were enrolled with parental or guardian consent. Stool samples were collected and subjected to culture and biochemical tests using standard protocols. Susceptibility testing was performed using conventional antibiotics. Among the participants (196), 125 (63.8%) were from pediatric outpatient departments, while 71 (36.2%) were from pediatric emergency departments. The study population consisted of 105 (53.6%) males and 91 (46.4%) females. Enteric bacterial pathogens were identified in 43 (21.9%) of the samples, with Escherichia coli accounting for 25 (12.8%), Shigella spp. for 11 (5.6%), and Salmonella spp. for 7 (3.6%). All isolated E. coli, Shigella spp., and Salmonella spp. exhibited susceptibility to Ceftriaxone. Notably, 14 (56%) and 13 (52%) of E. coli isolates demonstrated resistance to Amoxicillin and Ampicillin, respectively. The findings provide insights into the distribution of bacteria causing diarrhea in children under five, and associated antibiotic resistance profiles. The investigation highlights an urgent need for effective surveillance on bacterial pathogenicity among children below 5 years, antimicrobial stewardship, and alternative treatment strategies to combat the growing threat of multidrug-resistant enteric bacteria in this vulnerable population.
Title of Manuscript: Prevalence of SARS- CoV-2 virus in saliva, stool, and urine samples of COVID-19 patients in Bihar, India
Authors: Meghna Nupur , Archana Archana , Bhushan Divendu , Kumar Abhyuday , Sarfraz Asim , Naik Bijaya Nanda , Pati Binod Kumar
Published: August 2023

Introduction

The coronavirus illness caused by SARS- CoV-2 can cause multiple organ involvement, with varying degrees of severity. Besides, inhalation as a route for transmission, feco-oral has also been proposed. Its transmission to sewage systems is a growing public health issue.

Objective

To detect SARS-CoV-2 RNA in non-respiratory samples (saliva, urine, and stool) collected from COVID-19 cases, in Bihar.

Materials and methods

This Cross-Sectional observational study was conducted from January 2021 to March 2022 on human non-respiratory samples. A total of 345 samples including saliva (116), stool (97), and urine (132) were collected from 143 covid-19 cases. Samples were analyzed for SARS-CoV-2 by multiplex RT-PCR targeted against E, ORF 1ab, and RdRp gene.

Results

In this study, out of 143 cases, a total of 107(74.8%) were positive for SARS-CoV-2 RNA in at least one of the non-respiratory samples.

Conclusion

There is a high prevalence of SARS-CoV-2 virus in non-respiratory samples.
Flow: a web platform and open database to analyse, store, curate and share bioinformatics data at scale
Authors: Capitanchik Charlotte , Ireland Sam , Harston Alex , Cheshire Chris , Jones D. Marc , Lee Flora C.Y. , Ruiz de los Mozos Igor , Iosub Ira A. , Kuret Klara , Faraway Rupert , Wilkins Oscar G. , Arora Rahul , Hallegger Martina , Modic Miha , Chakrabarti Anob M. , Luscombe Nicholas M. , Ule Jernej
Published: August 2023

Ever-increasing volumes of sequencing data offer potential for large-scale meta-analyses to address significant biological questions. However, challenges such as insufficient data processing information, data quality concerns, and issues related to accessibility and curation often present obstacles. Additionally, most experimental biologists lack the time and expertise needed to independently analyse, manage and share their own data. To overcome these hurdles, we present Flow, a web-based platform that links bioinformatic analysis and database solutions with a user-friendly interface and web API. Flow currently accommodates a range of genomics methods and further DSL2-compliant Nextflow pipelines can be added via a simple JSON schema file. Deployable on local systems or cloud services, an instance is freely accessible to academic researchers athttps://flow.bio.
Inferring single-cell protein levels and cell cycle behavior in heterogeneous cell populations
Authors: Thijssen Bram , Segeren Hendrika A. , Liu Qingwu , Wessels Lodewyk F.A. , Westendorp Bart
Published: August 2023

Individual cells in a genetically identical population can show highly variable behavior. Single-cell measurements allow us to study this variability, but the available measurement techniques have limitations: live-cell microscopy is typically restricted to one or a few molecular markers, while techniques that simultaneously measure large numbers of molecular markers are destructive and cannot be used to follow cells over time. To help overcome these limitations, we present here scMeMo (single cell Mechanistic Modeler): a mechanistic modeling framework that can leverage diverse sets of measurements in order to infer unobserved variables in heterogeneous single cells. We used this framework to construct a model describing cell cycle progression in human cells, and show that it can predict the levels of several proteins in individual cells, based on live-cell microscopy measurements of only one marker and information learned from other experiments. The framework incorporates an uncertainty calibration step that makes the posterior distributions robust against partial model misspecification. Our modeling framework can be used to integrate information from separate experiments with diverse readouts, and to infer single cell variables that may be difficult to measure directly.
Gene editing ofSAMHD1in macrophage-like cells reveals complex relationships between SAMHD1 phospho-regulation, HIV-1 restriction and cellular dNTP levels
Authors: Schüssler Moritz , Schott Kerstin , Fuchs Nina Verena , Oo Adrian , Zahadi Morssal , Rauch Paula , Kim Baek , König Renate
Published: August 2023

Sterile α motif (SAM) and HD domain-containing protein 1 (SAMHD1) is a dNTP triphosphate triphosphohydrolase (dNTPase) and a potent restriction factor for immunodeficiency virus 1 (HIV-1), active in myeloid and resting CD4+T cells. The anti-viral activity of SAMHD1 is regulated by dephosphorylation of the residue T592. However, the impact of T592 phosphorylation on dNTPase activity is still under debate. Whether additional cellular functions of SAMHD1 impact anti-viral restriction is not completely understood.

We report BLaER1 cells as a novel human macrophage HIV-1 infection model combined with CRISPR/Cas9 knock-in (KI) introducing specific mutations into theSAMHD1locus to study mutations in a physiological context. Transdifferentiated BLaER1 cells harbor active dephosphorylated SAMHD1 that blocks HIV-1 reporter virus infection. As expected, homozygous T592E mutation, but not T592A, relieved a block to HIV-1 reverse transcription. Co-delivery of VLP-Vpx to SAMHD1 T592E KI mutant cells did not further enhance HIV-1 infection indicating the absence of an additional SAMHD1-mediated antiviral activity independent of T592 de-phosphorylation. T592E KI cells retained dNTP levels similar to WT cells indicating uncoupling of anti-viral and dNTPase activity of SAMHD1. The integrity of the catalytic site in SAMHD1 was critical for anti-viral activity, yet poor correlation of HIV-1 restriction and global cellular dNTP levels was observed in cells harboring catalytic core mutations. Together, we emphasize the complexity of the relationship between HIV-1 restriction, SAMHD1 enzymatic function and T592 phospho-regulation and provide novel tools for investigation in an endogenous and physiological context.

Importance

We introduce BLaER1 cells as an alternative myeloid cell model in combination with CRISPR/Cas9-mediated gene editing to study the influence of SAMHD1 T592 Mophosphorylation on anti-viral restriction and the control of cellular dNTP levels in an endogenous, physiological relevant context. Proper understanding of the mechanism of the anti-viral function of SAMHD1 will provide attractive strategies aiming at selectively manipulating SAMHD1 without affecting other cellular functions.

Even more, our toolkit may inspire further genetic analysis and investigation of restriction factors inhibiting retroviruses, their cellular function and regulation, leading to a deeper understanding of intrinsic anti-viral immunity.
Fe-TAMLs as a new class of small molecule peroxidase probes for correlated light and electron microscopy
Authors: Adams Stephen R. , Mackey Mason R. , Ramachandra Ranjan , Deerinck Thomas J. , Castillon Guillaume A. , Phan Sebastien , Hu Junru , Boassa Daniela , Ngo John T. , Ellisman Mark H.
Published: August 2023

We introduce Fe-TAML, a small molecule-based peroxidase as a versatile new member of the correlated fluorescence and electron microscopy toolkit. The utility of the probe is demonstrated by high resolution imaging of newly synthesized DNA (through biorthogonal labeling), genetically tagged proteins (using HaloTag), and untagged endogenous proteins (via immunostaining). EM visualization in these applications is facilitated by exploiting Fe-TAML’s catalytic activity for the deposition of localized osmiophilic precipitates based on polymerized 3,3’-diaminobenzidine. Optimized conditions for synthesizing and implementing Fe-TAML based probes are also described. Overall, Fe-TAML is a new chemical biology tool that can be used to visualize diverse biomolecular species along nanometer and micron scales within cells.
Structural mechanism of synergistic targeting of theCX3CR1nucleosome by PU.1 and C/EBP⍺
Authors: Lian Tengfei , Guan Ruifang , Zhou Bing-Rui , Bai Yawen
Published: August 2023

Pioneer transcription factors are vital for cell fate changes. PU.1 and C/EBP⍺ work together to regulate hematopoietic stem cell differentiation. However, how they recognizein vivonucleosomal DNA targets remain elusive. Here we report the structures of the nucleosome containing the mouse genomicCX3CR1enhancer DNA and its complexes with PU.1 alone and with both PU.1 and the C/EBP⍺ DNA binding domain. Our structures reveal that PU.1 binds the DNA motif at the exit linker, shifting 17 bp of DNA into the core region through interactions with H2A, unwrapping ∼20 bp of nucleosomal DNA. C/EBP⍺ binding, aided by PU.1’s repositioning, unwraps ∼25 bp entry DNA. The PU.1 Q218H mutation, linked to acute myeloid leukemia, disrupts PU.1-H2A interactions. PU.1 and C/EBP⍺ jointly displace linker histone H1 and open the H1-condensed nucleosome array. Our study unveils how two pioneer factors can work cooperatively to open closed chromatin by altering DNA positioning in the nucleosome.
Random walk and cell morphology dynamics inNaegleria gruberi
Authors: Uwamichi Masahito , Miura Yusuke , Kamiya Ayako , Imoto Daisuke , Sawai Satoshi
Published: August 2023

Amoeboid cell movement and migration are wide-spread across various cell types and species. Microscopy-based analysis of the model systemsDictyosteliumand neutrophils over the years have uncovered generality in their overall cell movement pattern. Under no directional cues, the centroid movement can be quantitatively characterized by their persistence to move in a straight line and the frequency of re-orientation. Mathematically, the cells essentially behave as a persistent random walker with memory of two characteristic time-scale. Such quantitative characterization is important from a cellular-level ethology point of view as it has direct connotation to their exploratory and foraging strategies. Interestingly, outside the amoebozoa and metazoa, there are largely uncharacterized species in the excavate taxon Heterolobosea including amoeboflagellateNaegleria. While classical works have shown that these cells indeed show typical amoeboid locomotion on an attached surface, their quantitative features are so far unexplored. Here, we analyzed the cell movement ofNaegleria gruberiby employing long-time phase contrast imaging to automatically track individual cells. We show that the cells move as a persistent random walker with two time-scales that are close to those known inDictyosteliumand neutrophils. Similarities were also found in the shape dynamics which is characterized by the appearance, splitting and annihilation of the curvature waves along the cell edge. Our analysis based on the Fourier descriptor and a neural network classifier point to importance of morphology features unique toNaegleriaincluding complex protrusions and the transient bipolar dumbbell morphologies.
Thyroid hormones mediate the impact of early-life stress on ventral tegmental area gene expression and behavior
Authors: Bennett Shannon N. , Chang Austin B. , Rogers Forrest D. , Jones Parker , Peña Catherine Jensen
Published: August 2023

Proper thyroid function is essential to the developing brain, including dopamine neuron differentiation, growth, and maintenance. Stress across the lifespan impacts thyroid hormone signaling and anxiety disorders and depression have been associated with thyroid dysfunction (both hypo- and hyper-active). However, less is known about how stress during postnatal development impacts thyroid function and related brain development. Our previous work in mice demonstrated that early-life stress (ELS) transiently impinged on expression of a transcription factor in dopamine neurons shown to be regulated by thyroid hormones. We hypothesized that thyroid hormone signaling may link experience of ELS with transcriptional dysregulation within the dopaminergic midbrain, and ultimately behavior. Here, we find that ELS transiently increases thyroid-stimulating hormone levels (inversely related to thyroid signaling) in both male and female mice at P21, an effect which recovers by adolescence. We next tested whether transient treatment of ELS mice with synthetic thyroid hormone (levothyroxine, LT4) could ameliorate the impact of ELS on sensitivity to future stress, and on expression of genes related to dopamine neuron development and maintenance, thyroid signaling, and plasticity within the ventral tegmental area. Among male mice, but not females, juvenile LT4 treatment prevented hypersensitivity to adult stress. We also found that rescuing developmental deficits in thyroid hormone signaling after ELS restored levels of some genes altered directly by ELS, and prevented alterations in expression of other genes sensitive to the second hit of adult stress. These findings suggest that thyroid signaling mediates the deleterious impact of ELS on VTA development, and that temporary treatment of hypothyroidism after ELS may be sufficient to prevent future stress hypersensitivity.
Hawkes process modelling for chemical reaction networks in a random environment
Authors: Sinzger-D’Angelo Mark , Koeppl Heinz
Published: August 2023

Cellular processes are open systems, situated in a heterogeneous context, rather than operating in isolation. Chemical reaction networks (CRNs) whose reaction rates are modelled as external stochastic processes account for the heterogeneous environment when describing the embedded process. A marginal description of the embedded process is of interest for (i) fast simulations that bypass the co-simulation of the environment, (ii) obtaining new process equations from which moment equations can be derived, (iii) the computation of information-theoretic quantities, and (iv) state estimation. It is known since Snyder’s and related works that marginalization over a stochastic intensity turns point processes into self-exciting ones. While the Snyder filter specifies the exact history-dependent propensities in the framework of CRNs in Markov environment, it was recently suggested to use approximate filters for the marginal description. By regarding the chemical reactions as events, we establish a link between CRNs in a linear random environment and Hawkes processes, a class of self-exciting counting processes widely used in event analysis. The Hawkes approximation can be obtained via moment closure scheme or as the optimal linear approximation under the quadratic criterion. We show the equivalence of both approaches. Furthermore, we use martingale techniques to provide results on the agreement of the Hawkes process and the exact marginal process in their second order statistics, i.e., covariance, auto/cross-correlation. We introduce an approximate marginal simulation algorithm and illustrate it in case studies.

AMS subject classifications

37M05, 60G35, 60G55, 60J28, 60K37, 62M15
Variable PD-1 glycosylation modulates the activity of immune checkpoint inhibitors
Authors: Chu Chih-Wei , Čaval Tomislav , Alisson-Silva Frederico , Tankasala Akshaya , Guerrier Christina , Czerwieniec Gregg , Läubli Heinz , Schwarz Flavio
Published: August 2023

Monoclonal antibodies targeting the immune checkpoint PD-1 have provided significant clinical benefit across a number of solid tumors, with differences in efficacy and toxicity profiles possibly related to their intrinsic molecular properties. Here, we report that camrelizumab and cemiplimab engage PD-1 through interactions with its fucosylated glycan. Using a combination of protein and cell glycoengineering, we demonstrate that the two antibodies bind preferentially to PD-1 with a core fucose at the asparagine N58 residue. We then provide evidence that the concentration of fucosylated PD-1 in the blood of non-small cell lung cancer patients varies across different stages of disease. This study illustrates how glycoprofiling of surface receptors and related circulating forms can inform the development of differentiated antibodies that discriminate glycosylation variants and achieve enhanced selectivity and paves the way towards the implementation of personalized therapeutic approaches.
From biological data to oscillator models using SINDy
Authors: Prokop Bartosz , Gelens Lendert
Published: August 2023

Periodic changes in the concentration or activity of different molecules regulate vital cellular processes such as cell division and circadian rhythms. Developing mathematical models is essential to better understand the mechanisms underlying these oscillations. Recent data-driven methods like SINDy have fundamentally changed model identification, yet their application to experimental biological data remains limited. This study investigates SINDy’s constraints by directly applying it to biological oscillatory data. We identify insufficient resolution, noise, dimensionality, and limited prior knowledge as primary limitations. Using various generic oscillator models of different complexity and/or dimensionality, we systematically analyze these factors. We then propose a comprehensive guide for inferring models from biological data, addressing these challenges step by step. Our approach is validated using glycolytic oscillation data from yeast.
Unravelling Genomic Origins of Lumpy Skin Disease Virus in Recent Outbreaks
Authors: Yadav Priya , Kumar Ankeet , Nath Sujith S , Shashidhar Geetha , Joshi Madhvi , Puvar Apurva , Sharma Sonal , Raval Janvi , Pandit Rameshchandra , Chavada Priyank , Nagaraj Sudheep , Revanaiah Yogesharadhya , Patil Deepak , Raval S K , Raval Jigar , Kanani Amit , Thakar Falguni , Kumar Naveen , Reddy Gundallhalli Bayyappa Manjunatha , Joshi Chaitanya , Gulati Baldev Raj , Tatu Utpal
Published: August 2023

Lumpy skin disease virus (LSDV) belongs to the genusCapripoxvirus and familyPoxviridae. LSDV was endemic in most of Africa, Middle east and Turkey, but since 2015, several outbreaks have been reported in Asian countries. In this study we used Whole Genome Sequence (WGS) approach to investigate the origin of the outbreak and understand the genomic landscape of the virus. Our study showed that the LSDV strain of 2022 outbreak exhibited many genetic variations, compared to the Reference Neethling strain sequence and the previous field strains from India. A total of 1819 variations were found in 22 genome sequences, which includes 399 extragenic mutations, 153 insertion frameshift mutations, 234 deletion frameshift mutations, 271 Single nucleotide polymorphisms (SNPs) and 762 silent SNPs. 38 genes have more than 2 variations per gene and these genes belong to viral-core protein, viral binding proteins, replication and RNA polymerase proteins. We highlight the importance of several SNPs in various genes which may play an essential role in pathogenesis of LSDV. Phylogenetic analysis performed on all whole genome sequences of LSDV showed two types of variants in India. One group of the variant with fewer mutations was found to lie closer to the LSDV 2019 strain from Ranchi while the other group clustered with previous Russian outbreaks from 2015. Our study highlights the importance of genomic characterization of viral outbreaks to not only monitor the frequency of mutations but also address its role in pathogenesis of LSDV as the outbreak continues.
Tools for Genetic Engineering and Gene Expression Control inNovosphingobium aromaticivoransandRhodobacter sphaeroides
Authors: Hall Ashley N. , Hall Benjamin W. , Kinney Kyle J. , Olsen Gabby G. , Banta Amy B. , Noguera Daniel R. , Donohue Timothy J. , Peters Jason M.
Published: August 2023

Alphaproteobacteria have a variety of cellular and metabolic features that provide important insights into biological systems and enable biotechnologies. For example, some species are capable of converting plant biomass into valuable biofuels and bioproducts have the potential to form the backbone of the sustainable bioeconomy. Among the Alphaproteobacteria,Novosphingobium aromaticivorans,Rhodobacter sphaeroides, andZymomonas mobilis, show particular promise as organisms that can be engineered to convert extracted plant lignin or sugars into bioproducts and biofuels. Genetic manipulation of these bacteria is needed to introduce engineered pathways and modulate expression of native genes with the goal of enhancing bioproduct output. Although recent work has expanded the genetic toolkit forZ. mobilis,N. aromaticivoransandR. sphaeroidesstill need facile, reliable approaches to deliver genetic payloads to the genome and to control gene expression. Here, we expand the platform of genetic tools forN. aromaticivoransandR. sphaeroidesto address these issues. We demonstrate that Tn7transposition is an effective approach for introducing engineered DNA into the chromosome ofN. aromaticivoransandR. sphaeroides. We screen a synthetic promoter library to identify inducible promoters with strong, regulated activity in both organisms. Combining Tn7integration with promoters from our library, we establish CRISPR interference systems forN. aromaticivoransandR. sphaeroidesthat can target essential genes and modulate engineered pathways. We anticipate that these systems will greatly facilitate both genetic engineering and gene function discovery efforts in these industrially important species and other Alphaproteobacteria.

IMPORTANCE

It is important to increase our understanding of the microbial world to improve health, agriculture, the environment and biotechnology. For example, building a sustainable bioeconomy depends on the efficient conversion of plant material to valuable biofuels and bioproducts by microbes. One limitation in this conversion process is that microbes with otherwise excellent properties for conversion are challenging to genetically engineer. Here, we report systems to overcome that barrier in the Alphaproteobacteria,Novosphingobium aromaticivoransandRhodobacter sphaeroides, by producing genetic tools that allow easy insertion of engineered pathways into their genomes and to precisely control gene expression by inducing genes with synthetic promoters or repressing genes using CRISPR interference. These tools can be used in future work to gain additional insight into these and other Alphaproteobacteria and to optimize yield of biofuels and bioproducts.
How Should Eukaryotic Chemotaxis be Measured?
Authors: Tweedy Luke , Thomason Peter A. , Insall Robert H.
Published: August 2023

Chemotaxis and directed cell migration are fundamentally important to eukaryotic biology. To understand the mechanisms that drive such complex processes, informative and robust measurements are essential, but the field does not always agree what these should be. Here we identify the most dependable measures of chemotactic steering and the underlying cell migration, and provide tools to test them. Some widely-used metrics can end up misleading, in particular “cos 8” for directional accuracy. We suggest that chemotactic efficiency should be used as a primary metric. Mean squared displacement and directional autocorrelation can be used to unpick different models of random and directed cell migration. Transition matrices are another useful tool for understanding migration mechanisms and avoiding artefacts, and provide a graphical illustration of how well cells maintain each direction. Unexpectedly, the choice of start and end points of tracks strongly affects the measurements and can seriously bias the measured results. This is particularly clear when cells are not homogeneously distributed at the start of an assay. To support straightforward adoption of these methods, we provide a suite of tools as a plugin for the open-source ImageJ program, and describe how they can be used to understand complex scenarios like self-generated chemotactic gradients.
CaMKII autophosphorylation but not downstream kinase activity is required for synaptic memory
Authors: Chen Xiumin , Cai Qixu , Zhou Jing , Pleasure Samuel J. , Schulman Howard , Zhang Mingjie , Nicoll Roger A.
Published: August 2023

CaMKII plays a critical role in long-term potentiation (LTP), a well-established model for learning and memory through the enhancement of synaptic transmission. Biochemical studies indicate that CaMKII catalyzes a phosphotransferase (kinase) reaction of both itself (autophosphorylation) and of multiple downstream target proteins. However, whether either type of phosphorylation plays any role in the synaptic enhancing action of CaMKII remains hotly contested. We have designed a series of experiments to define the minimal requirements for the synaptic enhancement by CaMKII. We find that autophosphorylation of T286 and further binding of CaMKII to the GluN2B subunit are required both for initiating LTP and for its maintenance (synaptic memory). Once bound to the NMDA receptor, the synaptic action of CaMKII occurs in the absence of kinase activity. Thus, autophosphorylation, together with binding to the GluN2B subunit, are the only two requirements for CaMKII in synaptic memory.
A glucanotransferase that uses two sub-sites and four catalytic aspartates/glutamates to disproportionate oligosaccharides ranging in length from maltotriose to starch
Authors: Sarkar Arpita , Kaila Pallavi , Tiwari Prince , Guptasarma Purnananda
Published: August 2023

PF0272 (PfuAmyGT) fromPyrococcus furiosusis a 656 residues-long, homodimeric, three-domain GH57 glycoside hydrolase [homologous to TLGT fromThermococcus litoralis(PDB ID: 1K1X)]. It is proposed to be either an α-amylase (EC 3.2.1.1), or a 4-α-glucanotransferase (EC 2.4.1.25). We demonstrate that PfuAmyGT is an exo-amylase-cum-glucanotransferase capable of transferring glucose, and dis-proportionating oligosaccharides, by excising glucose from malto-oligosaccharides (ranging in length from maltotriose to amylose/starch), and transferring it to malto-oligosaccharides (ranging in length from glucose to maltoheptaose and, possibly, even longer lengths). Convention holds that glucanotransferases transfer sugars through the serial and alternating binding of donors and acceptors to the same site, with covalent retention of excised sugars between such bindings. We present evidence of multiple behaviors in PfuAmyGT that challenge this view: (i) Production of free glucose, indicating scope for release of excised glucose; (ii) Higher activity with longer donors, indicating processivity; (iii) Accelerated activity with shorter acceptors, indicating a dependence upon rapid acceptor turnover; and (iv) Evidence of four catalytic glutamates/aspartates (E131, D222, E224, D362), indicating possible separation of excision and ligation functions. These behaviours collectively indicate the binding of donors and acceptors to separate sub-sites that have different substrate ‘length’ preferences, supporting our own previous proposal regarding separate tunnel (internal) and groove (surface) binding sites. Although PfuAmyGT’s mechanism of function remains to be fully elucidated, this paper definitively demonstrate ‘coupling’ of exo-amylase and glucanotransferase functions involving separate sub-sites for donor and acceptor binding.
Kinetic Proofreading can Enhance Specificity in a Non-enzymatic DNA Strand Displacement Network
Authors: Mukherjee Rakesh , Sengar Aditya , Cabello-García Javier , Ouldridge Thomas E.
Published: August 2023

Kinetic proofreading is used throughout natural systems to enhance the specificity of molecular recognition. At its most basic level, kinetic proofreading uses a supply of chemical fuel to drive a recognition interaction out of equilibrium, allowing a single free-energy difference between correct and incorrect targets to be exploited two or more times. Despite its importance in biology, there has been little effort to incorporate kinetic proofreading into synthetic systems in which molecular recognition is important, such as nucleic acid nanotechnology. In this article, we introduce a DNA strand displacement-based kinetic proofreading motif, showing that the consumption of a DNA-based fuel can be used to enhance molecular recognition during a templated dimerization reaction. We then show that kinetic proofreading can enhance the specificity with which a probe discriminates single nucleotide mutations, both in terms of the initial rate with which the probe reacts and the long-time behaviour.
Differences in the activities of domain-swapped chimeras of two homologous GH57 glucanotransferases suggest that a glucan-binding DUF could influence donor substrate specificity
Authors: Sarkar Arpita , Kaila Pallavi , Guptasarma Purnananda
Published: August 2023

Five enzymes of the archaeal hyperthermophilic family of disproportionating GH57 4-α-glucanotransferases have been studied till date. Our focus here lies upon three homologous members of this family: (i) PfuAmyGT fromPyrococcus furiosus(PF0272), (ii) TonAmyGT fromThermococcus onnurenius(B6YUX8), and (iii) TliAmyGT (TLGT) fromThermococcus litoralis(O32462). The polypeptide chain of each of these enzymes is approximately 655 residues long, folded into three distinct domains (1, 2 and 3), and assembled into a homodimer. Domain 1 is a beta/alpha barrel containing an aspartate known to function as a catalytic nucleophile in TLGT. Domain 2 (which is helical) and domain 3 (made up of beta sheets) are thought to be domains of unknown function (or DUFs). In PfuAmyGT and TonAmyGT, we have recently identified a catalytically-important aspartate upon a loop in domain 2. In PfuAmyGT, we demonstrate the presence of two additional catalytically-important (glutamate) residues in domain 1, in a companion paper. In this paper, our focus lies upon domain 3 which hosts a second binding site (SBS) for a glucan, at its domain-domain interface with domain 2. Using strategies involving studies of both (a) domains (or pairs of contiguous domains) extracted from PfuAmyGT, and (b) chimeric three-domain enzymes recombining analogous domains between PfuAmyGT and TonAmyGT, we demonstrate that domain 3 determines the choice of the preferred glucan that acts as a donor in the glucan transfer reaction.
Forensic Facial Approximation of Achondroplastic Dwarf from Medieval Cemetery in Central Europe
Authors: Moraes Cicero , Krenz-Niedbała Marta , Łukasik Sylwia , Prada Camilo Serrano
Published: August 2023

Achondroplasia (ACH, achondroplastic dwarfism) represents the most common form of skeletal dysplasia, occurring in c. 4 out of every 100,000 births. This study presents a computer-based facial approximation of the skull of a male individual suffering from ACH, who died at 30-45 years of age and was buried in Łekno, Poland between the 9th and 11th centuries AD. For the approximation procedure, soft tissue data from CT scans and ultrasonic measurements performed on living individuals were used. Additionally, the anatomical deformation technique was applied to arrive at the most reliable reconstruction of the dwarf’s appearance. To our knowledge, this is the first recreation of a person with achondroplasia, and one of the few showing a head of an individual suffering from a hereditary disease, with dimensions and shape differing from the population average values.

Highlights

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Forensic facial approximation of an achondroplastic dwarf from 9th–11th century AD has been performed as the first in the world

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The applied procedure included CT of a virtual donor

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Anatomical deformation technique allowed to extract the endocast, revealing a large volume of the endocranium

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Few measurements applied to the facial skeleton proved successful in identification of a person suffering from achondroplasia
An amplicon panel for high-throughput and low-cost genotyping of Pacific oyster
Authors: Sutherland Ben J. G. , Thompson Neil F. , Surry Liam B. , Gujjula Krishna Reddy , Carrasco Claudio D. , Chadaram Srinivas , Lunda Spencer L. , Langdon Christopher J. , Chan Amy M. , Suttle Curtis A. , Green Timothy J.
Published: August 2023

Maintaining genetic diversity in cultured shellfish can be challenging due to high variance in individual reproductive success, founder effects, and rapid genetic drift, but is important to retain adaptive potential and avoid inbreeding depression. To support broodstock management and selective breeding in cultured Pacific oysters (Crassostrea(Magallana)gigas), we developed an amplicon panel targeting 592 genomic regions and SNP variants with an average of 50 amplicons per chromosome. Target SNPs were selected based on elevated observed heterozygosity or differentiation in Pacific oyster populations in British Columbia (BC), Canada. The use of the panel for parentage applications was evaluated by genotyping three generations of oysters from a breeding program in BC (n = 181) and a set of families that were selected for Ostreid herpesvirus-1 (OSHV-1) resistance from the Molluscan Broodstock Program, a Pacific oyster breeding program in Oregon, USA (n = 136). Population characterization was evaluated using collections of wild, naturalized, farmed, or hatchery oysters sampled throughout the Northern Hemisphere (n = 190). Technical replicate samples showed high genotype concordance (97.5%; n = 68 replicates). Initial parentage analysis found instances of suspected pedigree and sample handling errors, demonstrating the panel’s value for quality control in breeding programs. Suspected null alleles were identified in parentage datasets and were found to reduce assignment success. Null alleles were largely population dependent, suggesting population-specific variation impacts target amplification. By taking an iterative approach, null alleles were identified using existing data without the need for pedigree information, and once null alleles were removed, assignments increased to 93.0% and 86.0% of possible assignments in the two breeding program datasets. A pipeline for analyzing the amplicon sequence data from sequencer output,amplitools, is also provided.
Unravelling ATP processing by the AAA+ protein p97 at the atomic level
Authors: Shein Mikhail , Hitzenberger Manuel , Cheng Tat Cheung , Rout Smruti R. , Leitl Kira D. , Sato Yusuke , Zacharias Martin , Sakata Eri , Schütz Anne K.
Published: August 2023

The human enzyme p97 regulates various cellular pathways by unfolding hundreds of protein substrates in an ATP-dependent manner, making it an essential component of homeostasis and impactful pharmacological target.

The hexameric complex undergoes substantial conformational changes in the course of its catalytic cycle. Here, we elucidate the molecular motions that occur at the active site in the temporal window immediately before and after ATP hydrolysis by merging cryo-EM, NMR spectroscopy and MD simulations. p97 populates a metastable reaction intermediate, the ADP.Pi state, which is poised between hydrolysis and product release. Detailed snapshots reveal that the active site is finely tuned to trap and eventually discharge the cleaved phosphate. Signalling pathways originating at the active site coordinate the action of the hexamer subunits and couple hydrolysis with allosteric conformational changes.

Our multidisciplinary approach enables a glimpse into the sophisticated spatial and temporal orchestration of ATP handling by a prototype AAA+ protein.
FRET-GP – A Local Measure of the Impact of Transmembrane Peptide on Lipids
Authors: Thakur Garima C. N. , Uday Arunima , Jurkiewicz Piotr
Published: August 2023

Reconstitution of a transmembrane protein in model lipid systems allows studying its structure and dynamics in isolation from the complexity of the natural environment. This approach also provides a well-defined environment for studying the interactions of the protein with lipids. In this work we describe the FRET-GP method, which utilizes Förster resonance energy transfer (FRET) to specifically probe nanoenvironment of a transmembrane domain. The tryptophan residues flanking this domain act as efficient FRET donors, while Laurdan acts as acceptor. The fluorescence of this solvatochromic probe, is quantified using generalized polarization (GP) to reports on lipid fluidity in the vicinity of the transmembrane domain. We applied FRET-GP to study the transmembrane peptide WALP incorporated in liposomes. We found that the direct excitation of Laurdan to its second singlet state strongly contribute to GP values measured in FRET conditions. Removal of this parasitic contribution was essential for proper determination ofGPFRET– the local analogue of classicalGPparameter. The presence of WALP significantly increased both parameters, but the local effects were considerably stronger (GPFRET≫GP). We conclude that WALP restricts lipid movement in its vicinity, inducing lateral inhomogeneity in membrane fluidity. WALP was also found to influence lipid phase transition. Our findings demonstrated that FRET-GP simultaneously provides local and global results, thereby increasing comprehensibility of the measurement. We highlight the simplicity and sensitivity of the method, but also discuss its potential and limitations in studying protein-lipid interactions.

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TRPM7 regulates phagocytosis and clearance ofCandida albicans
Authors: Stremska Marta E. , Stipes Eric J. , Jang Jessica J. , Busey Gregory W. , Iobst Wesley H. , Seegren Philip V. , Kennedy Joel , Desai Bimal N.
Published: August 2023

Sentinel phagocytes of the innate immune system have a critical role in detecting and eliminating fungal pathogens. We used patch clamp electrophysiology to explore the electrical signals elicited when macrophages engulfCandida albicans. In the perforated patch configuration, which is least disruptive to intracellular signaling, we detected a composite outwardly rectifying current during the engulfment ofC. albicansor zymosan. FTY720, a known inhibitor of ion channel TRPM7, suppressed the current. We then tested the hypothesis that TRPM7 regulates the engulfment and clearance ofC. albicans. We found thatTrpm7-/-macrophages are highly deficient in the engulfment ofC. albicans. Trpm7-/-macrophages initiate phagocytosis of yeast but are defective in sealing the phagocytic cups. While the precise mechanism through which TRPM7 regulates phagosome sealing is not clear, we tested the immunological significance of this discovery using a mouse model of systemic candidiasis. We show that in mice, wherein TRPM7 is deleted selectively in the myeloid cells, infection byC. albicansresults in significantly higher lethality, increased colonization of vital organs and increased inflammatory cytokines in the blood. Our study establishes TRPM7 as an ion channel critical for the innate immune responses against fungal pathogens and sets the stage for cell biological studies that define the mechanisms through which TRPM7 regulates phagosome sealing.

Significance statement

The worldwide increase in deadly or persistent fungal infections has prompted the research for alternative ways of treatment. We applied the specialized, perforated patch clamp technique to track and identify electrical currents elicited during the detection and engulfment of fungi by macrophages. The ion channel TRPM7 emerged as an important determinant of anti-fungal host defense as its deletion in the murine myeloid cells made the host mice highly susceptible to lethal candidiasis. Ion channels are attractive drug targets whose activation and inhibition can be manipulated with pharmacological therapeutics. This study raises the possibility of enhancing fungal clearance using activators of TRPM7. Such pharmacological strategy may benefit patients of persistent fungal infections that are recalcitrant to anti-fungal drugs.