A clinically attenuated double-mutant of porcine reproductive and respiratory syndrome virus-2 that does not prompt overexpression of proinflammatory cytokines during co-infection with a secondary pathogen.

Porcine reproductive and respiratory syndrome virus (PRRSV) is known to suppress the type I interferon (IFNs-α/ß) response during infection. PRRSV also activates the NF-κB signaling pathway, leading to the production of proinflammatory cytokines during infection. In swine farms, co-infections of PRRSV and other secondary bacterial pathogens are common and exacerbate the production of proinflammatory cytokines, contributing to the porcine respiratory disease complex (PRDC) which is clinically a severe disease. Previous studies identified the non-structural protein 1ß (nsp1ß) of PRRSV-2 as an IFN antagonist and the nucleocapsid (N) protein as the NF-κB activator. Further studies showed the leucine at position 126 (L126) of nsp1ß as the essential residue for IFN suppression and the region spanning the nuclear localization signal (NLS) of N as the NF-κB activation domain. In the present study, we generated a double-mutant PRRSV-2 that contained the L126A mutation in the nsp1ß gene and the NLS mutation (ΔNLS) in the N gene using reverse genetics. The immunological phenotype of this mutant PRRSV-2 was examined in porcine alveolar macrophages (PAMs) in vitro and in young pigs in vivo. In PAMs, the double-mutant virus did not suppress IFN-ß expression but decreased the NF-κB-dependent inflammatory cytokine productions compared to those for wild-type PRRSV-2. Co-infection of PAMs with the mutant PRRSV-2 and Streptococcus suis (S. suis) also reduced the production of NF-κB-directed inflammatory cytokines. To further examine the cytokine profiles and the disease severity by the mutant virus in natural host animals, 6 groups of pigs, 7 animals per group, were used for co-infection with the mutant PRRSV-2 and S. suis. The double-mutant PRRSV-2 was clinically attenuated, and the expressions of proinflammatory cytokines and chemokines were significantly reduced in pigs after bacterial co-infection. Compared to the wild-type PRRSV-2 and S. suis co-infection control, pigs coinfected with the double-mutant PRRSV-2 exhibited milder clinical signs, lower titers and shorter duration of viremia, and lower expression of proinflammatory cytokines. In conclusion, our study demonstrates that genetic modification of the type I IFN suppression and NF-κB activation functions of PRRSV-2 may allow us to design a novel vaccine candidate to alleviate the clinical severity of PRRS-2 and PRDC during bacterial co-infection.

Phosphorylation Mechanism Switching in Histidine Kinases Is a Tool for Fast Protein Evolution: Insights From AlphaFold Models.

Histidine kinases (HKs) are a central part of bacterial environmental-sensing two-component systems. They provide their hosts with the ability to respond to a wide range of physical and chemical signals. HKs are multidomain proteins consisting of at least a sensor domain, dimerization and phosphorylation domain (DHp), and a catalytic domain. They work as homodimers and the existence of two different autophosphorylation mechanisms (cis and trans) has been proposed as relevant for pathway specificity. Although several HKs have been intensively studied, a precise sequence-to-structure explanation of why and how either cis or trans phosphorylation occurs is still unavailable nor is there any evolutionary analysis on the subject. In this work, we show that AlphaFold can accurately determine whether an HK dimerizes in a cis or trans structure. By modeling multiple HKs we show that both cis- and trans-acting HKs are common in nature and the switch between mechanisms has happened multiple times in the evolutionary history of the family. We then use AlphaFold modeling to explore the molecular determinants of the phosphorylation mechanism. We conclude that it is the difference in lengths of the helices surrounding the DHp loop that determines the mechanism. We also show that very small changes in these helices can cause a mechanism switch. Despite this, previous evidence shows that for a particular HK the phosphorylation mechanism is conserved. This suggests that the phosphorylation mechanism participates in system specificity and mechanism switching provides these systems with a way to diverge.

Tumor mutational burden for the prediction of PD-(L)1 blockade efficacy in cancer: challenges and opportunities.

Tumor mutational burden (TMB) is a biomarker that measures the number of somatic mutations in a tumor's genome. TMB has emerged as a predictor of response to immune checkpoint inhibitors (ICIs) in various cancer types, and several studies have shown that patients with high TMB have better outcomes when treated with programmed death-ligand 1-based therapies. Recently, the Food and Drug Administration has approved TMB as a companion diagnostic for the use of pembrolizumab in solid tumors. However, despite its potential, the use of TMB as a biomarker for immunotherapy efficacy is limited by several factors. Here we review the limitations of TMB in predicting immunotherapy outcomes in patients with cancer and discuss potential strategies to optimize its use in the clinic.

Intrapatient variation in PD-L1 expression and tumor mutational burden and the impact on outcomes to immune checkpoint inhibitor therapy in patients with non-small cell lung cancer.

BACKGROUND: PD-L1 tumor proportion score (TPS) and tumor mutational burden (TMB) are key predictive biomarkers for immune checkpoint inhibitors (ICI) efficacy in non-small cell lung cancer (NSCLC). Data on their variation across multiple samples are limited. METHODS: Patients with NSCLC and multiple PD-L1 TPS and/or TMB assessments were included. Clinicopathologic and genomic data were analyzed according to PD-L1 and TMB variation. RESULTS: In total, 402 PD-L1 sample pairs and 413 TMB sample pairs were included. Concordance between pairs was moderate for PD-L1 (ρ=0.53, P<0.0001) and high for TMB (ρ=0.80, P<0.0001). Shorter time between biopsies correlated with higher concordance in PD-L1, but not in TMB. Major increases (ΔTPS≥+50%) and decreases (ΔTPS≤-50%) in PD-L1 were observed in 9.7% and 8.0% of cases, respectively. PD-L1, but not TMB, decreased with intervening ICI (P=0.02). Acquired copy number loss of CD274, PDCD1LG2, and JAK2 were associated with major decrease in PD-L1 (q<0.05). Among patients with multiple PD-L1 assessments before ICI, cases where all samples had a PD-L1 ≥1%, compared to cases with at least one sample with PD-L1 <1% and another with PD-L1 ≥1%, achieved improved objective response rate and progression-free survival (PFS). Among patients with at least one PD-L1 <1% and one ≥1% before ICI, cases where the most proximal sample was PD-L1 ≥1% had longer median PFS compared to cases where the most proximal PD-L1 was <1%. Among patients with multiple TMB assessments before ICI, patients with a TMB ≥10 mut/Mb based on the most recent assessment, as compared to those with a TMB <10 mut/Mb, achieved improved PFS and OS to ICI; instead, no differences were observed when patients were categorized using the oldest TMB assessment. CONCLUSION: Despite intrapatient concordance in PD-L1 and TMB, variation in these biomarkers can influence ICI outcomes, warranting consideration for reassessment prior to ICI initiation when feasible.

CoverageMaster: comprehensive CNV detection and visualization from NGS short reads for genetic medicine applications.

CoverageMaster (CoM) is a copy number variation (CNV) calling algorithm based on depth-of-coverage maps designed to detect CNVs of any size in exome [whole exome sequencing (WES)] and genome [whole genome sequencing (WGS)] data. The core of the algorithm is the compression of sequencing coverage data in a multiscale Wavelet space and the analysis through an iterative Hidden Markov Model. CoM processes WES and WGS data at nucleotide scale resolution and accurately detects and visualizes full size range CNVs, including single or partial exon deletions and duplications. The results obtained with this approach support the possibility for coverage-based CNV callers to replace probe-based methods such as array comparative genomic hybridization and multiplex ligation-dependent probe amplification in the near future.

Genetic mapping reveals new loci and alleles for flowering time and plant height using the double round-robin population of barley.

Flowering time and plant height are two critical determinants of yield potential in barley (Hordeum vulgare). Despite their role in plant physiological regulation, a complete overview of the genetic complexity of flowering time and plant height regulation in barley is still lacking. Using a double round-robin population originated from the crossings of 23 diverse parental inbred lines, we aimed to determine the variance components in the regulation of flowering time and plant height in barley as well as to identify new genetic variants by single and multi-population QTL analyses and allele mining. Despite similar genotypic variance, we observed higher environmental variance components for plant height than flowering time. Furthermore, we detected new QTLs for flowering time and plant height. Finally, we identified a new functional allelic variant of the main regulatory gene Ppd-H1. Our results show that the genetic architecture of flowering time and plant height might be more complex than reported earlier and that a number of undetected, small effect, or low-frequency genetic variants underlie the control of these two traits.

Exploiting the Reactivity of Metal Trifluoroacetates to Access Alkali-Niobium(V) Oxyfluorides.

Motivated by the lack of facile routes to alkali-niobium(V) oxyfluorides KNb2O5F and CsNb2O5F, we investigated the reactivity of alkali trifluoroacetates KH(tfa)2 and CsH(tfa)2 (tfa = CF3COO-) toward Nb2O5 in the solid state. Tetragonal tungsten bronze KNb2O5F and pyrochlore CsNb2O5F were obtained by simply reacting the corresponding trifluoroacetate with Nb2O5 at 600 °C under air, without the need for specialized containers or a controlled atmosphere. Thermolysis of KH(tfa)2 in the presence of Nb2O5 yielded single-phase polycrystalline KNb2O5F. By contrast, the reaction between CsH(tfa)2 and Nb2O5 produced a mixture of CsNb2O5F and a new oxyfluoride of formula CsNb3O7F2, whose crystal structure was solved using powder X-ray and electron diffraction. CsNb3O7F2 (space group P6/mmm) belongs to the family of hexagonal tungsten bronzes and features an open-framework structure consisting of corner-sharing Nb(O,F)6 octahedra with hexagonal channels occupied by Cs+ ions. Isomorphous RbNb3O7F2 was obtained upon reacting RbH(tfa)2 with Nb2O5. Synthetic optimization enabled the preparation of RbNb3O7F2 and CsNb3O7F2 as single-phase polycrystalline solids at 500 °C under flowing synthetic air. Both oxyfluorides were found to be semiconductors with a band gap of ≈3.5 eV. The discovery of these two oxyfluorides highlights the importance of probing the reactivity of solids whose full potential as fluorinated precursors is yet to be realized.

Dominant monoallelic variant in the PAK2 gene causes Knobloch syndrome type 2.

Knobloch syndrome is an autosomal recessive phenotype mainly characterized by retinal detachment and encephalocele caused by biallelic pathogenic variants in the COL18A1 gene. However, there are patients clinically diagnosed as Knobloch syndrome with unknown molecular etiology not linked to COL18A1. We studied an historical pedigree (published in 1998) designated as KNO2 (Knobloch type 2 syndrome with intellectual disability, autistic behavior, retinal degeneration, encephalocele). Whole exome sequencing of the two affected siblings and the normal parents resulted in the identification of a PAK2 non-synonymous substitution p.(Glu435Lys) as a causative variant. The variant was monoallelic and apparently de novo in both siblings indicating a likely germ-line mosaicism in one of the parents; the mosaicism, however, could not be observed after deep sequencing of blood parental DNA. PAK2 encodes a member of a small group of serine/threonine kinases; these P21-activating kinases (PAKs) are essential in signal transduction and cellular regulation (cytoskeletal dynamics, cell motility, death and survival signaling and cell cycle progression). Structural analysis of the PAK2 p.(Glu435Lys) variant that is located in the kinase domain of the protein predicts a possible compromise in the kinase activity. Functional analysis of the p.(Glu435Lys) PAK2 variant in transfected HEK293T cells results in a partial loss of the kinase activity. PAK2 has been previously suggested as an autism-related gene. Our results show that PAK2-induced phenotypic spectrum is broad and not fully understood. We conclude that the KNO2 syndrome in the studied family is dominant and caused by a deleterious variant in the PAK2 gene.

De Novo and Bi-allelic Pathogenic Variants in NARS1 Cause Neurodevelopmental Delay Due to Toxic Gain-of-Function and Partial Loss-of-Function Effects.

Aminoacyl-tRNA synthetases (ARSs) are ubiquitous, ancient enzymes that charge amino acids to cognate tRNA molecules, the essential first step of protein translation. Here, we describe 32 individuals from 21 families, presenting with microcephaly, neurodevelopmental delay, seizures, peripheral neuropathy, and ataxia, with de novo heterozygous and bi-allelic mutations in asparaginyl-tRNA synthetase (NARS1). We demonstrate a reduction in NARS1 mRNA expression as well as in NARS1 enzyme levels and activity in both individual fibroblasts and induced neural progenitor cells (iNPCs). Molecular modeling of the recessive c.1633C>T (p.Arg545Cys) variant shows weaker spatial positioning and tRNA selectivity. We conclude that de novo and bi-allelic mutations in NARS1 are a significant cause of neurodevelopmental disease, where the mechanism for de novo variants could be toxic gain-of-function and for recessive variants, partial loss-of-function.

Clonal KEAP1 mutations with loss of heterozygosity share reduced immunotherapy efficacy and low immune cell infiltration in lung adenocarcinoma.

BACKGROUND: KEAP1 mutations have been associated with reduced survival in lung adenocarcinoma (LUAD) patients treated with immune checkpoint inhibitors (ICIs), particularly in the presence of STK11/KRAS alterations. We hypothesized that, beyond co-occurring genomic events, clonality prediction may help identify deleterious KEAP1 mutations and their counterparts with retained sensitivity to ICIs. PATIENTS AND METHODS: Beta-binomial modelling of sequencing read counts was used to infer KEAP1 clonal inactivation by combined somatic mutation and loss of heterozygosity (KEAP1 C-LOH) versus partial inactivation [KEAP1 clonal diploid-subclonal (KEAP1 CD-SC)] in the Memorial Sloan Kettering Cancer Center (MSK) MetTropism cohort (N = 2550). Clonality/LOH prediction was compared to a streamlined clinical classifier that relies on variant allele frequencies (VAFs) and tumor purity (TP) (VAF/TP ratio). The impact of this classification on survival outcomes was tested in two independent cohorts of LUAD patients treated with immunotherapy (MSK/Rome N = 237; DFCI N = 461). Immune-related features were studied by exploiting RNA-sequencing data (TCGA) and multiplexed immunofluorescence (DFCI mIF cohort). RESULTS: Clonality/LOH inference in the MSK MetTropism cohort overlapped with a clinical classification model defined by the VAF/TP ratio. In the ICI-treated MSK/Rome discovery cohort, predicted KEAP1 C-LOH mutations were associated with shorter progression-free survival (PFS) and overall survival (OS) compared to KEAP1 wild-type cases (PFS log-rank P = 0.001; OS log-rank P < 0.001). Similar results were obtained in the DFCI validation cohort (PFS log-rank P = 0.006; OS log-rank P = 0.014). In both cohorts, we did not observe any significant difference in survival outcomes when comparing KEAP1 CD-SC and wild-type tumors. Immune deconvolution and multiplexed immunofluorescence revealed that KEAP1 C-LOH and KEAP1 CD-SC differed for immune-related features. CONCLUSIONS: KEAP1 C-LOH mutations are associated with an immune-excluded phenotype and worse clinical outcomes among advanced LUAD patients treated with ICIs. By contrast, survival outcomes of patients whose tumors harbored KEAP1 CD-SC mutations were similar to those with KEAP1 wild-type LUADs.

Spatial and Seasonal Variations in the Bacterial Community of an Anthropogenic Impacted Urban Stream.

Environmental changes and human activities can alter the structure and diversity of aquatic microbial communities. In this work, we analyzed the bacterial community dynamics of an urban stream to understand how these factors affect the composition of river microbial communities. Samples were taken from a stream situated in Buenos Aires, Argentina, which flows through residential, peri-urban horticultural, and industrial areas. For sampling, two stations were selected: one influenced by a series of industrial waste treatment plants and horticultural farms (PL), and the other influenced by residential areas (R). Microbial communities were analyzed by sequence analysis of 16S rRNA gene amplicons along an annual cycle. PL samples showed high nutrient content compared with R samples. The diversity and richness of the R site were more affected by seasonality than those of the PL site. At the amplicon sequence variants level, beta diversity analysis showed a differentiation between cool-season (fall and winter) and warm-season (spring and summer) samples, as well as between PL and R sites. This demonstrated that there is spatial and temporal heterogeneity in the composition of the bacterial community, which should be considered if a bioremediation strategy is applied. The taxonomic composition analysis also revealed a differential seasonal cycle of phototrophs and chemoheterotrophs between the sampling sites, as well as different taxa associated with each sampling site. This analysis, combined with a comparative analysis of global rivers, allowed us to determine the genera Arcobacter, Simplicispira, Vogesella, and Sphingomonas as potential bioindicators of anthropogenic disturbance.

Bacillus-Based Direct-Fed Microbial Reduces the Pathogenic Synergy of a Coinfection with Salmonella enterica Serovar Choleraesuis and Porcine Reproductive and Respiratory Syndrome Virus.

Viral respiratory infections predispose lungs to bacterial coinfections causing a worse outcome than either infection alone. Porcine reproductive and respiratory syndrome virus (PRRSV) causes pneumonia in pigs and is often associated with bacterial coinfections. We examined the impact of providing weanling pigs a Bacillus-based direct-fed microbial (DFM) on the syndrome resulting from infection with either Salmonella enterica serotype Choleraesuis alone, or in combination with PRRSV. Nine days after the bacterial challenge, Salmonella was isolated from ileocecal lymph nodes of all challenged pigs regardless of DFM treatment. Compared to the single bacterial challenge, the dual challenge with Salmonella and PRRSV resulted in a pathogenic synergy exhibited by a higher rate of Salmonella colonization in the lung and a more extensive and severe interstitial pneumonia. Provision of DFM to dually challenged pigs reduced the rate of lung colonization by Salmonella, eliminated or reduced the presence of PRRSV in the lung, and reduced the extent and severity of gross lung pathology. Dually challenged pigs that received DFM had increased concentrations of interleukin 1 (IL-1) and IL-8 in lung lavage fluids, accompanied by increased expression in their blood cells of nucleotide-binding oligomerization domain receptor 2 (NOD2) and triggering receptor expressed in myeloid cells 1 (TREM-1) molecules. These changes in pulmonary inflammatory cytokine production and increased expression of NOD2 and TREM-1 suggest that the DFM exerted a systemic modulating effect on innate immunity. These observations are consistent with the notion that tonic stimulation by gut-derived microbial products can poise innate immunity to fight infections in the respiratory tract.

Biallelic variants in PSMB1 encoding the proteasome subunit ß6 cause impairment of proteasome function, microcephaly, intellectual disability, developmental delay and short stature.

The molecular cause of the majority of rare autosomal recessive disorders remains unknown. Consanguinity due to extensive homozygosity unravels many recessive phenotypes and facilitates the detection of novel gene-disease links. Here, we report two siblings with phenotypic signs, including intellectual disability (ID), developmental delay and microcephaly from a Pakistani consanguineous family in which we have identified homozygosity for p(Tyr103His) in the PSMB1 gene (Genbank NM_002793) that segregated with the disease phenotype. PSMB1 encodes a ß-type proteasome subunit (i.e. ß6). Modeling of the p(Tyr103His) variant indicates that this variant weakens the interactions between PSMB1/ß6 and PSMA5/α5 proteasome subunits and thus destabilizes the 20S proteasome complex. Biochemical experiments in human SHSY5Y cells revealed that the p(Tyr103His) variant affects both the processing of PSMB1/ß6 and its incorporation into proteasome, thus impairing proteasome activity. CRISPR/Cas9 mutagenesis or morpholino knock-down of the single psmb1 zebrafish orthologue resulted in microcephaly, microphthalmia and reduced brain size. Genetic evidence in the family and functional experiments in human cells and zebrafish indicates that PSMB1/ß6 pathogenic variants are the cause of a recessive disease with ID, microcephaly and developmental delay due to abnormal proteasome assembly.

Taurine treatment of retinal degeneration and cardiomyopathy in a consanguineous family with SLC6A6 taurine transporter deficiency.

In a consanguineous Pakistani family with two affected individuals, a homozygous variant Gly399Val in the eighth transmembrane domain of the taurine transporter SLC6A6 was identified resulting in a hypomorph transporting capacity of ~15% compared with normal. Three-dimensional modeling of this variant has indicated that it likely causes displacement of the Tyr138 (TM3) side chain, important for transport of taurine. The affected individuals presented with rapidly progressive childhood retinal degeneration, cardiomyopathy and almost undetectable plasma taurine levels. Oral taurine supplementation of 100 mg/kg/day resulted in maintenance of normal blood taurine levels. Following approval by the ethics committee, a long-term supplementation treatment was introduced. Remarkably, after 24-months, the cardiomyopathy was corrected in both affected siblings, and in the 6-years-old, the retinal degeneration was arrested, and the vision was clinically improved. Similar therapeutic approaches could be employed in Mendelian phenotypes caused by the dysfunction of the hundreds of other molecular transporters.

Bi-allelic Variants in DYNC1I2 Cause Syndromic Microcephaly with Intellectual Disability, Cerebral Malformations, and Dysmorphic Facial Features.

Cargo transport along the cytoplasmic microtubular network is essential for neuronal function, and cytoplasmic dynein-1 is an established molecular motor that is critical for neurogenesis and homeostasis. We performed whole-exome sequencing, homozygosity mapping, and chromosomal microarray studies in five individuals from three independent pedigrees and identified likely-pathogenic variants in DYNC1I2 (Dynein Cytoplasmic 1 Intermediate Chain 2), encoding a component of the cytoplasmic dynein 1 complex. In a consanguineous Pakistani family with three affected individuals presenting with microcephaly, severe intellectual disability, simplification of cerebral gyration, corpus callosum hypoplasia, and dysmorphic facial features, we identified a homozygous splice donor site variant (GenBank: NM_001378.2:c.607+1G>A). We report two additional individuals who have similar neurodevelopmental deficits and craniofacial features and harbor deleterious variants; one individual bears a c.740A>G (p.Tyr247Cys) change in trans with a 374 kb deletion encompassing DYNC1I2, and an unrelated individual harbors the compound-heterozygous variants c.868C>T (p.Gln290∗) and c.740A>G (p.Tyr247Cys). Zebrafish larvae subjected to CRISPR-Cas9 gene disruption or transient suppression of dync1i2a displayed significantly altered craniofacial patterning with concomitant reduction in head size. We monitored cell death and cell cycle progression in dync1i2a zebrafish models and observed significantly increased apoptosis, likely due to prolonged mitosis caused by abnormal spindle morphology, and this finding offers initial insights into the cellular basis of microcephaly. Additionally, complementation studies in zebrafish demonstrate that p.Tyr247Cys attenuates gene function, consistent with protein structural analysis. Our genetic and functional data indicate that DYNC1I2 dysfunction probably causes an autosomal-recessive microcephaly syndrome and highlight further the critical roles of the dynein-1 complex in neurodevelopment.

Bi-allelic Variants in IQSEC1 Cause Intellectual Disability, Developmental Delay, and Short Stature.

We report two consanguineous families with probands that exhibit intellectual disability, developmental delay, short stature, aphasia, and hypotonia in which homozygous non-synonymous variants were identified in IQSEC1 (GenBank: NM_001134382.3). In a Pakistani family, the IQSEC1 segregating variant is c.1028C>T (p.Thr343Met), while in a Saudi Arabian family the variant is c.962G>A (p.Arg321Gln). IQSEC1-3 encode guanine nucleotide exchange factors for the small GTPase ARF6 and their loss affects a variety of actin-dependent cellular processes, including AMPA receptor trafficking at synapses. The ortholog of IQSECs in the fly is schizo and its loss affects growth cone guidance at the midline in the CNS, also an actin-dependent process. Overexpression of the reference IQSEC1 cDNA in wild-type flies is lethal, but overexpression of the two variant IQSEC1 cDNAs did not affect viability. Loss of schizo caused embryonic lethality that could be rescued to 2nd instar larvae by moderate expression of the human reference cDNA. However, the p.Arg321Gln and p.Thr343Met variants failed to rescue embryonic lethality. These data indicate that the variants behave as loss-of-function mutations. We also show that schizo in photoreceptors is required for phototransduction. Finally, mice with a conditional Iqsec1 deletion in cortical neurons exhibited an increased density of dendritic spines with an immature morphology. The phenotypic similarity of the affecteds and the functional experiments in flies and mice indicate that IQSEC1 variants are the cause of a recessive disease with intellectual disability, developmental delay, and short stature, and that axonal guidance and dendritic projection defects as well as dendritic spine dysgenesis may underlie disease pathogenesis.

Methylated tetra-amide derivatives of paramagnetic complexes for magnetic resonance biosensing with both BIRDS and CEST.

Paramagnetic agents that utilize two mechanisms to provide physiological information by magnetic resonance imaging (MRI) and magnetic resonance spectroscopic imaging (MRSI) are described. MRI with chemical exchange saturation transfer (CEST) takes advantage of the agent's exchangeable protons (e.g., -OH or -NHx , where 2 ≥ x ≥ 1) to create pH contrast. The agent's incorporation of non-exchangeable protons (e.g., -CHy , where 3 ≥ y ≥ 1) makes it possible to map tissue temperature and/or pH using an MRSI method called biosensor imaging of redundant deviation in shifts (BIRDS). Hybrid probes based upon 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate chelate (DOTA4- ) and its methylated analog (1,4,7,10-tetraazacyclododecane-α, α', α″, α‴-tetramethyl-1,4,7,10-tetraacetate, DOTMA4- ) were synthesized, and modified to create new tetra-amide chelates. Addition of several methyl groups per pendent arm of the symmetrical chelates, positioned proximally and distally to thulium ions (Tm3+ ), gave rise to favorable BIRDS properties (i.e., high signal-to-noise ratio (SNR) from non-exchangeable methyl proton peaks) and CEST responsiveness (i.e., from amide exchangeable protons). Structures of the Tm3+ probes elucidate the influence of methyl group placement on sensor performance. An eight-coordinate geometry with high symmetry was observed for the complexes: Tm-L1 was based on DOTA4- , whereas Tm-L2 and Tm-L3 were based on DOTMA4- , where the latter contained an additional carboxylate at the distal end of each arm. The distance of Tm3+ from terminal methyl carbons is a key determinant for sustaining BIRDS temperature sensitivity without compromising CEST pH contrast; however, water solubility was influenced by introduction of hydrophobic methyl groups and hydrophilic carboxylate. Combined BIRDS and CEST detection of Tm-L2, which features two high-SNR methyl peaks and a strong amide CEST peak, should enable simultaneous temperature and pH measurements for high-resolution molecular imaging in vivo.

The double round-robin population unravels the genetic architecture of grain size in barley.

Grain number, size and weight primarily determine the yield of barley. Although the genes regulating grain number are well studied in barley, the genetic loci and the causal gene for sink capacity are poorly understood. Therefore, the primary objective of our work was to dissect the genetic architecture of grain size and weight in barley. We used a multi-parent population developed from a genetic cross between 23 diverse barley inbreds in a double round-robin design. Seed size-related parameters such as grain length, grain width, grain area and thousand-grain weight were evaluated in the HvDRR population comprising 45 recombinant inbred line sub-populations. We found significant genotypic variation for all seed size characteristics, and observed 84% or higher heritability across four environments. The quantitative trait locus (QTL) detection results indicate that the genetic architecture of grain size is more complex than previously reported. In addition, both cultivars and landraces contributed positive alleles at grain size QTLs. Candidate genes identified using genome-wide variant calling data for all parental inbred lines indicated overlapping and potential novel regulators of grain size in cereals. Furthermore, our results indicated that sink capacity was the primary determinant of grain weight in barley.

Urinary volatile Organic compounds in non-alcoholic fatty liver disease (NAFLD), type two diabetes mellitus (T2DM) and NAFLD-T2DM coexistence.

INTRODUCTION: Accumulating evidence have shown a significant correlation between urinary volatile organic compounds (VOCs) profile and the manifestation of several physiological and pathological states, including liver diseases. Previous studies have investigated the urinary metabolic signature as a non-invasive tool for the early discrimination between non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH), which nowadays represents one of the most important challenges in this context, feasible only by carrying out liver biopsy. OBJECTIVES: The aim of the study was to investigate the differences in the urinary VOCs profiles of non-alcoholic fatty liver disease (NAFLD) patients, diabetes mellitus (T2DM) subjects and NAFLD/T2DM patients. METHODS: Headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS) was applied to profile the urinary VOCs. Urine samples were analysed both under acid and alkaline conditions, to obtain a range of urinary volatiles with different physicochemical properties. RESULTS: Urinary VOCs profiles of 13 NAFLD patients, 13 T2DM subjects and 13 NAFLD/T2DM patients were investigated by multivariate and univariate data analysis techniques which allowed to identify 21 volatiles under alkaline conditions able to describe the NAFLD/T2DM group concerning the other two groups. CONCLUSION: Our results suggest that VOCs signatures can improve the knowledge of the pathological condition where NAFLD coexists with T2DM and discovering new features that are not simply the sum of the two diseases. These preliminary findings may be considered as hypothesis-generating, to be clearly confirmed by larger prospective investigations.

Lithifying and Non-Lithifying Microbial Ecosystems in the Wetlands and Salt Flats of the Central Andes.

The wetlands and salt flats of the Central Andes region are unique extreme environments as they are located in high-altitude saline deserts, largely influenced by volcanic activity. Environmental factors, such as ultraviolet (UV) radiation, arsenic content, high salinity, low dissolved oxygen content, extreme daily temperature fluctuation, and oligotrophic conditions, resemble the early Earth and potentially extraterrestrial conditions. The discovery of modern microbialites and microbial mats in the Central Andes during the past decade has increased the interest in this area as an early Earth analog. In this work, we review the current state of knowledge of Central Andes region environments found within lakes, small ponds or puquios, and salt flats of Argentina, Chile, and Bolivia, many of them harboring a diverse range of microbial communities that we have termed Andean Microbial Ecosystems (AMEs). We have integrated the data recovered from all the known AMEs and compared their biogeochemistry and microbial diversity to achieve a better understanding of them and, consequently, facilitate their protection.