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The size and complexity of datasets resulting from comparative research experiments in the agricultural domain is constantly increasing. Often the number of variables measured in an experiment exceeds the number of experimental units composing the experiment. When there is a necessity to model the covariance relationships that exist between variables in these experiments, estimation difficulties can arise due to the resulting covariance structure being of reduced rank. A statistical method, based in a linear mixed model framework, is presented for the analysis of designed experiments where datasets are characterised by a greater number of variables than experimental units, and for which the modelling of complex covariance structures between variables is desired. Aided by a clustering algorithm, the method enables the estimation of covariance through the introduction of covariance clusters as random effects into the modelling framework, providing an extension of the traditional variance components model for building covariance structures. The method was applied to a multi-phase mass spectrometry-based proteomics experiment, with the aim of exploring changes in the proteome of barley grain over time during the malting process. The modelling approach provides a new linear mixed model-based method for the estimation of covariance structures between variables measured from designed experiments, when there are a small number of experimental units, or observations, informing covariance parameter estimates. [ABSTRACT FROM AUTHOR]

The ability of Mycobacterium tuberculosis (Mtb) to persist in the host complicates and prolongs tuberculosis (TB) patient chemotherapy. Here we demonstrate that a neglected two-component system (TCS) of Mtb, TcrXY, is an autoregulated acid-sensing TCS that controls a functionally diverse 70-gene regulon required for bacterial persistence. Characterisation of two representatives of this regulon, Rv3706c and Rv3705A, implicate these genes as key determinants for the survival of Mtb in vivo by serving as important effectors to mitigate redox stress at acidic pH. We show that genetic silencing of the response regulator tcrX using CRISPR interference attenuates the persistence of Mtb during chronic mouse infection and improves treatment with the two front-line anti-TB drugs, rifampicin and isoniazid. We propose that targeting TcrXY signal transduction blocks the ability of Mtb to sense and respond to acid stress, resulting in a disordered program of persistence to render the organism vulnerable to existing TB chemotherapy.Stupar et al. describe a new role for the Mycobacterium tuberculosis two-component system, TcrXY, in the modulation of up to 70 genes, including two effectors, TarA and TarB which mitigate intracellular redox stress. [ABSTRACT FROM AUTHOR]

In August 2022, a novel henipavirus (HNV) named Langya virus (LayV) was isolated from patients with severe pneumonic disease in China. This virus is closely related to Mòjiāng virus (MojV), and both are divergent from the bat-borne HNV members, Nipah (NiV) and Hendra (HeV) viruses. The spillover of LayV is the first instance of a HNV zoonosis to humans outside of NiV and HeV, highlighting the continuing threat this genus poses to human health. In this work, we determine the prefusion structures of MojV and LayV F proteins via cryogenic electron microscopy to 2.66 and 3.37 Å, respectively. We show that despite sequence divergence from NiV, the F proteins adopt an overall similar structure but are antigenically distinct as they do not react to known antibodies or sera. Glycoproteomic analysis revealed that while LayV F is less glycosylated than NiV F, it contains a glycan that shields a site of vulnerability previously identified for NiV. These findings explain the distinct antigenic profile of LayV and MojV F, despite the extent to which they are otherwise structurally similar to NiV. Our results carry implications for broad-spectrum HNV vaccines and therapeutics, and indicate an antigenic, yet not structural, divergence from prototypical HNVs. In 2022, the zoonotic Langya virus caused cases of severe pneumonia in humans. Here, the authors determine the structure of the fusion glycoprotein of Langya virus and demonstrate antigenic but not structural divergence from bat-borne henipaviruses, yielding insights for future vaccine designs. [ABSTRACT FROM AUTHOR]

The fundament of an evidence-based severity assessment in laboratory animal science is reliable distress parameters. Many readouts are used to evaluate and determine animal distress and the severity of experimental procedures. Therefore, we analyzed four distinct parameters like the body weight, burrowing behavior, nesting, and distress score in the four gastrointestinal animal models (pancreatic ductal adenocarcinoma (PDA), pancreatitis, CCl4 intoxication, and bile duct ligation (BDL)). Further, we determined the parameters' robustness in various experimental subgroups due to slight variations like drug treatment or telemeter implantations. We used non-parametric bootstrapping to get robust estimates and 95% confidence intervals for the experimental groups. It was found that the performance of the readout parameters is model-dependent and that the distress score is prone to experimental variation. On the other hand, we also found that burrowing and nesting can be more robust than, e.g., the body weight when evaluating PDA. However, the body weight still was highly robust in BDL, pancreatitis, and CCl4 intoxication. To address the complex nature of the multi-dimensional severity space, we used the Relative Severity Assessment (RELSA) procedure to combine multiple distress parameters into a score and mapped the subgroups and models against a defined reference set obtained by telemeter implantation. This approach allowed us to compare the severity of individual animals in the experimental subgroups using the maximum achieved severity (RELSAmax). With this, the following order of severity was found for the animal models: CCl4 < PDA ≈ Pancreatitis < BDL. Furthermore, the robustness of the RELSA procedure and outcome was externally validated with a reference set from another laboratory also obtained from telemeter implantation. Since the RELSA procedure reflects the multi-dimensional severity information and is highly robust in estimating the quantitative severity within and between models, it can be deemed a valuable tool for laboratory animal severity assessment. [ABSTRACT FROM AUTHOR]

Main conclusion: When gene editing was applied to knockout beta-kafirin, there was a compensatory increase of gamma-kafirin which does not occur in domesticated null varieties, so enhanced grain quality was not achieved. Sorghum bicolor is an important animal feedstock cereal crop throughout Australia and the southern United States, where its use as a food product is limited by issues with low calorific and nutritive value. Qualities such as reduced digestibility and low essential amino acid content are directly attributed to the kafirin grain storage proteins, the major components of protein bodies within the endosperm. Specifically, the β- and γ-kafirins have few protease cleavage sites and high levels of cysteine residues which lead to a highly cross-linked shell of intra- and inter-molecular disulphide linkages that encapsulate the more digestible α- and δ-kafirins in the core of the protein bodies. Naturally occurring β-kafirin mutants exist and are known to have improved grain quality, with enhanced protein contents and digestibility, traits which are often attributed to the lack of this cysteine-rich kafirin in the mature grain. However, when CRISPR/Cas9 editing was used to create β-kafirin knockout lines, there was no improvement to grain quality in the Tx430 background, although they did have unique protein composition and changes to protein body morphology in the vitreous endosperm. One explanation of the divergence in quality traits found the lines lacking β-kafirin are due to a drastic increase of γ-kafirin which was only found in the gene edited lines. This study highlights that in some germplasm, there is a level of redundancy between the peripheral kafirins, and that improvement of grain protein digestibility cannot be achieved by simply removing the β-kafirin protein in all genetic backgrounds. [ABSTRACT FROM AUTHOR]

Beer is one of the most popular beverages worldwide. As a product of variable agricultural ingredients and processes, beer has high molecular complexity. We used DIA/SWATH-MS to investigate the proteomic complexity and diversity of 23 commercial Australian beers. While the overall complexity of the beer proteome was modest, with contributions from barley and yeast proteins, we uncovered a very high diversity of post-translational modifications (PTMs), especially proteolysis, glycation, and glycosylation. Proteolysis was widespread throughout barley proteins, but showed clear site-specificity. Oligohexose modifications were common on lysines in barley proteins, consistent with glycation by maltooligosaccharides released from starch during malting or mashing. O-glycosylation consistent with oligomannose was abundant on secreted yeast glycoproteins. We developed and used data analysis pipelines to efficiently extract and quantify site-specific PTMs from SWATH-MS data, and showed incorporating these features into proteomic analyses extended analytical precision. We found that the key differentiator of the beer glyco/proteome was the brewery, with beer from independent breweries having a distinct profile to beer from multinational breweries. Within a given brewery, beer styles also had distinct glyco/proteomes. Targeting our analyses to beers from a single brewery, Newstead Brewing Co., allowed us to identify beer style-specific features of the glyco/proteome. Specifically, we found that proteins in darker beers tended to have low glycation and high proteolysis. Finally, we objectively quantified features of foam formation and stability, and showed that these quality properties correlated with the concentration of abundant surface-active proteins from barley and yeast. [ABSTRACT FROM AUTHOR]

Mosquito vectors transmit various diseases through blood feeding, required for their egg development. Hence, blood feeding is a major physiological event in their life cycle, during which hundreds of genes are tightly regulated. Blood is a rich source of proteins for mosquitoes, but also contains many other molecules including microRNAs (miRNAs). Here, we found that human blood miRNAs are transported abundantly into the fat body tissue of Aedes aegypti, a key metabolic center in post-blood feeding reproductive events, where they target and regulate mosquito genes. Using an artificial diet spiked with the mimic of an abundant and stable human blood miRNA, hsa-miR-21-5p, and proteomics analysis, we found over 40 proteins showing differential expression in female Ae. aegypti mosquitoes after feeding. Of interest, we found that the miRNA positively regulates the vitellogenin gene, coding for a yolk protein produced in the mosquito fat body and then transported to the ovaries as a protein source for egg production. Inhibition of hsa-miR-21-5p followed by human blood feeding led to a statistically insignificant reduction in progeny production. The results provide another example of the involvement of small regulatory molecules in the interaction of taxonomically vastly different taxa. Perdomo et al. fed mosquitos (Aedes aegypti) a diet spiked with human blood miRNA, hsa-miR-21-5p and used proteomics analysis to show differential protein expression in female Ae. aegypti mosquitoes after feeding. They showed that hsa-miR-21-5p positively regulated vitellogenin, which plays a role in egg production, thus could have effects on Ae. aegypti progeny production. [ABSTRACT FROM AUTHOR]

Coagulation factor IX (FIX) is a complex post-translationally modified human serum glycoprotein and high-value biopharmaceutical. The quality of recombinant FIX (rFIX), especially complete γ-carboxylation, is critical for rFIX clinical efficacy. Bioreactor operating conditions can impact rFIX production and post-translational modifications (PTMs). With the goal of optimizing rFIX production, we developed a suite of Data Independent Acquisition Mass Spectrometry (DIA-MS) proteomics methods and used these to investigate rFIX yield, γ-carboxylation, other PTMs, and host cell proteins during bioreactor culture and after purification. We detail the dynamics of site-specific PTM occupancy and structure on rFIX during production, which correlated with the efficiency of purification and the quality of the purified product. We identified new PTMs in rFIX near the GLA domain which could impact rFIX GLA-dependent purification and function. Our workflows are applicable to other biologics and expression systems, and should aid in the optimization and quality control of upstream and downstream bioprocesses. Zacchi et al. attempt to bridge the gap between understanding the impact of process variables and determination of critical post-translational modifications on a highly complex glycoprotein, factor IX. They implement an LC-MS/MS approach based on data independent acquisition which allows measurement of all detectable peptides, even with heterogeneous modifications. [ABSTRACT FROM AUTHOR]

Brewing science is undergoing a renaissance with the use of modern analytical chemistry and microbiology techniques. However, these modern analytical tools and techniques are not necessarily aligned with the scale and scope of brewing science. In particular, brewing processes can be time consuming, ingredient intensive, and require specialised technical equipment. These drawbacks compound with the need for appropriate numbers of replicates for adequately powered experimental design. Here, we describe a micro-scale mash method that can be performed using a common laboratory benchtop shaker/incubator, allowing for high throughput mashing and easy sample replication for statistical analysis. Proteomic profiles at both the protein and peptide levels were consistent between the 1 mL micro-mash and a 23 L Braumeister mash, and both mash scales produced wort with equivalent fermentable sugar and free amino acid profiles. The experimental flexibility offered by our micro-mash method allowed us to investigate the effects of altered mash parameters on the beer brewing proteome. [ABSTRACT FROM AUTHOR]

Human Factor IX is a highly post-translationally modified protein that is an important clotting factor in the blood coagulation cascade. Functional deficiencies in Factor IX result in the bleeding disorder haemophilia B, which is treated with plasma-derived or recombinant Factor IX concentrates. Here, we investigated the post-translational modifications of human serum-derived Factor IX and report previously undescribed O-linked monosaccharide compositions at serine 141 and a novel site of glycosylation. At serine 141 we observed two monosaccharide compositions, with HexNAc1Hex1NeuAc2 dominant and a low level of HexNAc1Hex1NeuAc1. This O-linked site lies N-terminal to the first cleavage site for the activation peptide, an important region of the protein that is removed to activate Factor IX. The novel site is an N-linked site in the serine protease domain with low occupancy in a non-canonical consensus motif at asparagine 258, observed with a HexNAc4Hex5NeuAc2 monosaccharide composition attached. This is the first reported instance of a site of modification in the serine protease domain. The description of these glycosylation events provides a basis for future functional studies and contributes to structural characterisation of native Factor IX for the production of effective therapeutic biosimilars and biobetters. [ABSTRACT FROM AUTHOR]

We analyzed the population genetic pattern of 12 fragmented Geropogon hybridus ecological range edge populations in Israel along a steep precipitation gradient. In the investigation area (45 × 20 km), the annual mean precipitation changes rapidly from 450 mm in the north (Mediterranean-influenced climate zone) to 300 mm in the south (semiarid climate zone) without significant temperature changes. Our analysis (91 individuals, 12 populations, 123 polymorphic loci) revealed strongly structured populations (AMOVA Φ = 0.35; P < 0.001); however, differentiation did not change gradually toward range edge. IBD was significant (Mantel test r = 0.81; P = 0.001) and derived from sharply divided groups between the northernmost populations and the others further south, due to dispersal or environmental limitations. This was corroborated by the PCA and STRUCTURE analyses. IBD and IBE were significant despite the micro-geographic scale of the study area, which indicates that reduced precipitation toward range edge leads to population genetic divergence. However, this pattern diminished when the hypothesized gene flow barrier was taken into account. Applying the spatial analysis method revealed 11 outlier loci that were correlated to annual precipitation and, moreover, were indicative for putative precipitation-related adaptation (BAYESCAN, MCHEZA). The results suggest that even on micro-geographic scales, environmental factors play prominent roles in population divergence, genetic drift, and directional selection. The pattern is typical for strong environmental gradients, e.g., at species range edges and ecological limits, and if gene flow barriers and mosaic-like structures of fragmented habitats hamper dispersal. [ABSTRACT FROM AUTHOR]

Glycosylation is a co- and post-translational modification that is critical for the regulation of the biophysical properties and biological activities of diverse proteins. Biosynthetic pathways for protein glycosylation are inherently inefficient, resulting in high structural diversity in mature glycoproteins. Macroheterogeneity is the structural diversity due to the presence or absence of glycans at specific glycosylation sites, and is caused by inefficiency in the initial transfer of glycans to proteins. Here, we review the enzymatic and evolutionary mechanisms controlling macroheterogeneity, its biological consequences in physiological and disease states, its relevance to heterologous production and glycoengineering of glycoproteins, and mass spectrometry based methods for its analysis. We highlight the importance of the analysis of macroheterogeneity for a complete understanding of glycoprotein biosynthesis and function, and emphasize how advances in mass spectrometry glycoproteomics will enable analysis of this critical facet of glycoprotein structural diversity. [ABSTRACT FROM AUTHOR]

Liver glycogen (involved in maintaining blood-sugar levels) is a hyperbranched glucose polymer containing β particles (diameter ~20 nm), which can form composite α particles (diameter ~50-300 nm), and includes a small but significant amount of bound protein. Size distributions of glycogen from livers of healthy and diabetic mice were examined using size-exclusion chromatography with two separate eluents: aqueous eluent and dimethylsulfoxide (DMSO) eluent. Morphologies were examined with transmission electron microscopy. Diabetic glycogen (DG) exhibited many α particles in the mild water-based solvent, but in DMSO, which breaks H bonds, these degraded to β particles; α particles however were always present in healthy glycogen (HG). This DG fragility shows the binding of β into α particles is different in HG and DG. The diabetic α particle fragility may be involved with the uncontrolled blood-sugar release symptomatic of diabetes: small β particles degrade more easily to glucose than α particles. This has implications for diabetes management. [ABSTRACT FROM AUTHOR]

The calculation of hydrogen positions is a common preprocessing step when working with crystal structures of protein-ligand complexes. An explicit description of hydrogen atoms is generally needed in order to analyze the binding mode of particular ligands or to calculate the associated binding energies. Due to the large number of degrees of freedom resulting from different chemical moieties and the high degree of mutual dependence this problem is anything but trivial. In addition to an efficient algorithm to take care of the complexity resulting from complicated hydrogen bonding networks, a robust chemical model is needed to describe effects such as tautomerism and ionization consistently. We present a novel method for the placement of hydrogen coordinates in protein-ligand complexes which takes tautomers and protonation states of both protein and ligand into account. Our method generates the most probable hydrogen positions on the basis of an optimal hydrogen bonding network using an empirical scoring function. The high quality of our results could be verified by comparison to the manually adjusted Astex diverse set and a remarkably low rate of undesirable hydrogen contacts compared to other tools. [ABSTRACT FROM AUTHOR]

The measurements of plasma natriuretic peptides (NT-proBNP, proBNP and BNP) are used to diagnose heart failure but these are expensive to produce. We describe a rapid, cheap and facile production of proteins for immunoassays of heart failure. DNA encoding N-terminally His-tagged NT-proBNP and proBNP were cloned into the pJexpress404 vector. ProBNP and NT-proBNP peptides were expressed in Escherichia coli, purified and refolded in vitro. The analytical performance of these peptides were comparable with commercial analytes (NT-proBNP EC for the recombinant is 2.6 ng/ml and for the commercial material is 5.3 ng/ml) and the EC for recombinant and commercial proBNP, are 3.6 and 5.7 ng/ml respectively). Total yield of purified refolded NT-proBNP peptide was 1.75 mg/l and proBNP was 0.088 mg/l. This approach may also be useful in expressing other protein analytes for immunoassay applications. Purpose of work: To develop a cost effective protein expression method in E. coli to obtain high yields of NT-proBNP (1.75 mg/l) and proBNP (0.088 mg/l) peptides for immunoassay use. [ABSTRACT FROM AUTHOR]

Systematic studies of thermotropic liquid crystals in confinement, such as liquid crystals in microfluidic channels, require control of the anchoring conditions on the surfaces. Especially for the case of uniform planar anchoring, the standard method involves a mechanical treatment (rubbing) of the surface that is not applicable to microfluidic devices. In the present study, we report methods for the achievement of well-defined anchoring conditions for liquid crystals in microfluidic channels consisting of polydimethylsiloxane and glass. Various physico-chemical techniques enable to establish homeotropic, degenerate planar, uniform planar, and hybrid anchoring conditions on the surface of the channel walls. We characterize the treated surfaces in terms of wettability and liquid crystal anchoring and determine the director field in the microchannels for the different anchoring configurations using polarizing optical microscopy and fluorescence confocal polarization microscopy. The relevance of the surface anchoring for the flow behavior of the liquid crystal in the microchannel is demonstrated by studying the onset of defect-mediated chaotic-like flow at high Ericksen numbers for the different anchoring cases. [ABSTRACT FROM AUTHOR]

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We describe a new method for producing homogeneous eukaryotic N-glycoproteins. The method involves the engineering and functional transfer of the Campylobacter jejuni glycosylation machinery in Escherichia coli to express glycosylated proteins with the key GlcNAc-Asn linkage. The bacterial glycans were then trimmed and remodeled in vitro by enzymatic transglycosylation to fulfill a eukaryotic N-glycosylation. It provides a potentially general platform for producing eukaryotic N-glycoproteins. [ABSTRACT FROM AUTHOR]

The re-emergence of scarlet fever poses a new global public health threat. The capacity of North-East Asian serotype M12 (emm12) Streptococcus pyogenes (group A Streptococcus, GAS) to cause scarlet fever has been linked epidemiologically to the presence of novel prophages, including prophage ΦHKU.vir encoding the secreted superantigens SSA and SpeC and the DNase Spd1. Here, we report the molecular characterization of ΦHKU.vir-encoded exotoxins. We demonstrate that streptolysin O (SLO)-induced glutathione efflux from host cellular stores is a previously unappreciated GAS virulence mechanism that promotes SSA release and activity, representing the first description of a thiol-activated bacterial superantigen. Spd1 is required for resistance to neutrophil killing. Investigating single, double and triple isogenic knockout mutants of the ΦHKU.vir-encoded exotoxins, we find that SpeC and Spd1 act synergistically to facilitate nasopharyngeal colonization in a mouse model. These results offer insight into the pathogenesis of scarlet fever-causing GAS mediated by prophage ΦHKU.vir exotoxins. The pathogenesis of Streptococcus pyogenes (GAS) causing scarlet fever has been associated with the presence of prophages, such as ΦHKU.vir, and their products. Here, the authors characterize the exotoxins SpeC and Spd1 of ΦHKU.vir and show these to act synergistically to facilitate nasopharyngeal colonization in mice. [ABSTRACT FROM AUTHOR]

The accumulation of advanced glycation end products (AGEs) have been implicated in the development and progression of diabetic kidney disease (DKD). There has been interest in investigating the potential of AGE clearance receptors, such as oligosaccharyltransferase-48 kDa subunit (OST48) to prevent the detrimental effects of excess AGE accumulation seen in the diabetic kidney. Here the objective of the study was to increase the expression of OST48 to examine if this slowed the development of DKD by facilitating the clearance of AGEs. Groups of 8-week-old heterozygous knock-in male mice (n = 9–12/group) over-expressing the gene encoding for OST48, dolichyl-diphosphooligosaccharide-protein glycosyltransferase (DDOST+/−) and litter mate controls were randomised to either (i) no diabetes or (ii) diabetes induced via multiple low-dose streptozotocin and followed for 24 weeks. By the study end, global over expression of OST48 increased glomerular OST48. This facilitated greater renal excretion of AGEs but did not affect circulating or renal AGE concentrations. Diabetes resulted in kidney damage including lower glomerular filtration rate, albuminuria, glomerulosclerosis and tubulointerstitial fibrosis. In diabetic mice, tubulointerstitial fibrosis was further exacerbated by global increases in OST48. There was significantly insulin effectiveness, increased acute insulin secretion, fasting insulin concentrations and AUCinsulin observed during glucose tolerance testing in diabetic mice with global elevations in OST48 when compared to diabetic wild-type littermates. Overall, this study suggested that despite facilitating urinary-renal AGE clearance, there were no benefits observed on kidney functional and structural parameters in diabetes afforded by globally increasing OST48 expression. However, the improvements in insulin secretion seen in diabetic mice with global over-expression of OST48 and their dissociation from effects on kidney function warrant future investigation. [ABSTRACT FROM AUTHOR]

Virulence of Cryptococcus neoformans is regulated by a range of transcription factors, and is also influenced by the acquisition of adaptive mutations during infection. Beyond the temporal regulation of virulence factor production by transcription factors and these permanent microevolutionary changes, heritable epigenetic modifications such as histone deacetylation may also play a role during infection. Here we describe the first comprehensive analysis of the sirtuin class of NAD+ dependent histone deacetylases in the phylum Basidiomycota, identifying five sirtuins encoded in the C. neoformans genome. Each sirtuin gene was deleted and a wide range of phenotypic tests performed to gain insight into the potential roles they play. Given the pleiotropic nature of sirtuins in other species, it was surprising that only two of the five deletion strains revealed mutant phenotypes in vitro. However, cryptic consequences of the loss of each sirtuin were identified through whole cell proteomics, and mouse infections revealed a role in virulence for SIR2, HST3 and HST4. The most intriguing phenotype was the repeated inability to complement mutant phenotypes through the reintroduction of the wild-type gene. These data support the model that regulation of sirtuin activity may be employed to enable a drastic alteration of the epigenetic landscape and virulence of C. neoformans. [ABSTRACT FROM AUTHOR]

An abstract of the article "A flexible-hydrogen interaction model for protein-ligand docking," by Angela M. Henzler, Sascha Urbaczek, Benjamin Schulz, and Matthias Rarey is presented.