A germline PAF1 paralog complex ensures cell type-specific gene expression.

Animal germline development and fertility rely on paralogs of general transcription factors that recruit RNA polymerase II to ensure cell type-specific gene expression. It remains unclear whether gene expression processes downstream from such paralog-based transcription is distinct from that of canonical RNA polymerase II genes. In Drosophila, the testis-specific TBP-associated factors (tTAFs) activate over a thousand spermatocyte-specific gene promoters to enable meiosis and germ cell differentiation. Here, we show that efficient termination of tTAF-activated transcription relies on testis-specific paralogs of canonical polymerase-associated factor 1 complex (PAF1C) proteins, which form a testis-specific PAF1C (tPAF). Consequently, tPAF mutants show aberrant expression of hundreds of downstream genes due to read-in transcription. Furthermore, tPAF facilitates expression of Y-linked male fertility factor genes and thus serves to maintain spermatocyte-specific gene expression. Consistently, tPAF is required for the segregation of meiotic chromosomes and male fertility. Supported by comparative in vivo protein interaction assays, we provide a mechanistic model for the functional divergence of tPAF and the PAF1C and identify transcription termination as a developmentally regulated process required for germline-specific gene expression.

Extended lactations in dairy cows and the effects on fertility and production.

Extending lactation length reduces the frequency of critical calving events for the cow and herewith reduces the frequency of periods with increased risk for health problems. Moreover, breeding is postponed until a moment later in lactation, which is associated with better conception rates and less days open after start of the breeding period in most studies. Potential risks of an extended lactation are that milk yield of cows at the end of the lactation may be too low which may lead to cows being overconditioned at the end of the extended lactation. Therefore, extending lactation length might not fit every cow. Individual cow characteristics like parity, milk yield level, or body condition determine the response of the cow to an extended lactation. These individual cow characteristics can be used in customized management strategies to optimize lactation length for individual cows. Customized lactation length for individual cows could limit the impact at herd level of disadvantages concerning milk losses and overconditioning and maintain benefits for improved cow health and fertility, reduced number of surplus calves and increased work satisfaction for the farmer. In conclusion, extending lactation length has interesting perspectives for health and fertility of high-producing dairy cows, although questions remain concerning management approaches to support lactation persistency of cows with an extended lactation, and consequences for calf health and development. Moreover, ongoing studies aim to develop decision support tools to select individual cows for a specific lactation length.

Oligomeric State of ß-Coronavirus Non-Structural Protein 10 Stimulators Studied by Small Angle X-ray Scattering.

The ß-coronavirus family, encompassing Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Severe Acute Respiratory Syndrome Coronavirus (SARS), and Middle East Respiratory Syndrome Coronavirus (MERS), has triggered pandemics within the last two decades. With the possibility of future pandemics, studying the coronavirus family members is necessary to improve knowledge and treatment. These viruses possess 16 non-structural proteins, many of which play crucial roles in viral replication and in other vital functions. One such vital protein is non-structural protein 10 (nsp10), acting as a pivotal stimulator of nsp14 and nsp16, thereby influencing RNA proofreading and viral RNA cap formation. Studying nsp10 of pathogenic coronaviruses is central to unraveling its multifunctional roles. Our study involves the biochemical and biophysical characterisation of full-length nsp10 from MERS, SARS and SARS-CoV-2. To elucidate their oligomeric state, we employed a combination of Multi-detection Size exclusion chromatography (Multi-detection SEC) with multi-angle static light scattering (MALS) and small angle X-ray scattering (SAXS) techniques. Our findings reveal that full-length nsp10s primarily exist as monomers in solution, while truncated versions tend to oligomerise. SAXS experiments reveal a globular shape for nsp10, a trait conserved in all three coronaviruses, although MERS nsp10, diverges most from SARS and SARS-CoV-2 nsp10s. In summary, unbound nsp10 proteins from SARS, MERS, and SARS-CoV-2 exhibit a globular and predominantly monomeric state in solution.

Coping with darkness: The adaptive response of marine picocyanobacteria to repeated light energy deprivation.

The picocyanobacteria Prochlorococcus and Synechococcus are found throughout the ocean's euphotic zone, where the daily light:dark cycle drives their physiology. Periodic deep mixing events can, however, move cells below this region, depriving them of light for extended periods of time. Here, we demonstrate that members of these genera can adapt to tolerate repeated periods of light energy deprivation. Strains kept in the dark for 3 d and then returned to the light initially required 18-26 d to resume growth, but after multiple rounds of dark exposure they began to regrow after only 1-2 d. This dark-tolerant phenotype was stable and heritable; some cultures retained the trait for over 132 generations even when grown in a standard 13:11 light:dark cycle. We found no genetic differences between the dark-tolerant and parental strains of Prochlorococcus NATL2A, indicating that an epigenetic change is likely responsible for the adaptation. To begin to explore this possibility, we asked whether DNA methylation-one potential mechanism mediating epigenetic inheritance in bacteria-occurs in Prochlorococcus. LC-MS/MS analysis showed that while DNA methylations, including 6 mA and 5 mC, are found in some other Prochlorococcus strains, there were no methylations detected in either the parental or dark-tolerant NATL2A strains. These findings suggest that Prochlorococcus utilizes a yet-to-be-determined epigenetic mechanism to adapt to the stress of extended light energy deprivation, and highlights phenotypic heterogeneity as an additional dimension of Prochlorococcus diversity.

In-depth Spatiotemporal Characterization of Planktonic Archaeal and Bacterial Communities in North and South San Francisco Bay.

Despite being the largest estuary on the west coast of North America, no in-depth survey of microbial communities in San Francisco Bay (SFB) waters currently exists. In this study, we analyze bacterioplankton and archaeoplankton communities at several taxonomic levels and spatial extents (i.e., North versus South Bay) to reveal patterns in alpha and beta diversity. We assess communities using high-throughput sequencing of the 16S rRNA gene in 177 water column samples collected along a 150-km transect over a 2-year monthly time-series. In North Bay, the microbial community is strongly structured by spatial salinity changes while in South Bay seasonal variations dominate community dynamics. Along the steep salinity gradient in North Bay, we find that operational taxonomic units (OTUs; 97% identity) have higher site specificity than at coarser taxonomic levels and turnover ("species" replacement) is high, revealing a distinct brackish community (in oligo-, meso-, and polyhaline samples) from fresh and marine end-members. At coarser taxonomic levels (e.g., phylum, class), taxa are broadly distributed across salinity zones (i.e., present/abundant in a large number of samples) and brackish communities appear to be a mix of fresh and marine communities. We also observe variations in brackish communities between samples with similar salinities, likely related to differences in water residence times between North and South Bay. Throughout SFB, suspended particulate matter is positively correlated with richness and influences changes in beta diversity. Within several abundant groups, including the SAR11 clade (comprising up to 30% of reads in a sample), OTUs appear to be specialized to a specific salinity range. Some other organisms also showed pronounced seasonal abundance, including Synechococcus, Ca. Actinomarina, and Nitrosopumilus-like OTUs. Overall, this study represents the first in-depth spatiotemporal survey of SFB microbial communities and provides insight into how planktonic microorganisms have specialized to different niches along the salinity gradient.

Crystal Structure of Non-Structural Protein 10 from Severe Acute Respiratory Syndrome Coronavirus-2.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), causing Coronavirus Disease 19 (COVID-19), emerged at the end of 2019 and quickly spread to cause a global pandemic with severe socio-economic consequences. The early sequencing of its RNA genome revealed its high similarity to SARS, likely to have originated from bats. The SARS-CoV-2 non-structural protein 10 (nsp10) displays high sequence similarity with its SARS homologue, which binds to and stimulates the 3'-to-5' exoribonuclease and the 2'-O-methlytransferase activities of nsps 14 and 16, respectively. Here, we report the biophysical characterization and 1.6 Å resolution structure of the unbound form of nsp10 from SARS-CoV-2 and compare it to the structures of its SARS homologue and the complex-bound form with nsp16 from SARS-CoV-2. The crystal structure and solution behaviour of nsp10 will not only form the basis for understanding the role of SARS-CoV-2 nsp10 as a central player of the viral RNA capping apparatus, but will also serve as a basis for the development of inhibitors of nsp10, interfering with crucial functions of the replication-transcription complex and virus replication.

Distance decay relationships in foliar fungal endophytes are driven by rare taxa.

Foliar fungal endophytes represent a diverse and species-rich plant microbiome. Their biogeography provides essential clues to their cryptic relationship with hosts and the environment in which they disperse. We present species composition, diversity, and dispersal patterns of endophytic fungi associated with needles of Pinus taeda trees across regional scales in the absence of strong environmental gradients as well as within individual trees. An empirical designation of rare and abundant taxa enlightens us on the structure of endophyte communities. We report multiple distance-decay patterns consistent with effects of dispersal limitation, largely driven by community changes in rare taxa, those taxonomic units that made up less than 0.31% of reads per sample on average. Distance-decay rates and community structure also depended on specific classes of fungi and were predominantly influenced by rare members of Dothideomycetes. Communities separated by urban areas also revealed stronger effects of distance on community similarity, confirming that host density and diversity plays an important role in symbiont biogeography, which may ultimately lead to a mosaic of functional diversity as well as rare species diversity across landscapes.

Global expression analysis of the yeast Lachancea (Saccharomyces) kluyveri reveals new URC genes involved in pyrimidine catabolism.

Pyrimidines are important nucleic acid precursors which are constantly synthesized, degraded, and rebuilt in the cell. Four degradation pathways, two of which are found in eukaryotes, have been described. One of them, the URC pathway, has been initially discovered in our laboratory in the yeast Lachancea kluyveri. Here, we present the global changes in gene expression in L. kluyveri in response to different nitrogen sources, including uracil, uridine, dihydrouracil, and ammonia. The expression pattern of the known URC genes, URC1-6, helped to identify nine putative novel URC genes with a similar expression pattern. The microarray analysis provided evidence that both the URC and PYD genes are under nitrogen catabolite repression in L. kluyveri and are induced by uracil or dihydrouracil, respectively. We determined the function of URC8, which was found to catalyze the reduction of malonate semialdehyde to 3-hydroxypropionate, the final degradation product of the pathway. The other eight genes studied were all putative permeases. Our analysis of double deletion strains showed that the L. kluyveri Fui1p protein transported uridine, just like its homolog in Saccharomyces cerevisiae, but we demonstrated that is was not the only uridine transporter in L. kluyveri. We also showed that the L. kluyveri homologs of DUR3 and FUR4 do not have the same function that they have in S. cerevisiae, where they transport urea and uracil, respectively. In L. kluyveri, both of these deletion strains grew normally on uracil and urea.

Microglia and motor neurons during disease progression in the SOD1G93A mouse model of amyotrophic lateral sclerosis: changes in arginase1 and inducible nitric oxide synthase.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting the motor system. Although the etiology of the disease is not fully understood, microglial activation and neuroinflammation are thought to play a role in disease progression. METHODS: We examined the immunohistochemical expression of two markers of microglial phenotype, the arginine-metabolizing enzymes inducible nitric oxide synthase (iNOS) and arginase1 (Arg1), in the spinal cord of a mouse model carrying an ALS-linked mutant human superoxide dismutase transgene (SOD1(G93A)) and in non-transgenic wild-type (WT) mice. Immunolabeling for iNOS and Arg1 was evaluated throughout disease progression (6 to 25 weeks), and correlated with body weight, stride pattern, wire hang duration and ubiquitin pathology. For microglia and motor neuron counts at each time point, SOD1(G93A) and WT animals were compared using an independent samples t-test. A Welch t-test correction was applied if Levene's test showed that the variance in WT and SOD1G93A measurements was substantially different. RESULTS: Disease onset, measured as the earliest change in functional parameters compared to non-transgenic WT mice, occurred at 14 weeks of age in SOD1(G93A) mice. The ventral horn of the SOD1(G93A) spinal cord contained more microglia than WT from 14 weeks onwards. In SOD1(G93A) mice, Arg1-positive and iNOS-positive microglia increased 18-fold and 7-fold, respectively, between 10 and 25 weeks of age (endpoint) in the lumbar spinal cord, while no increase was observed in WT mice. An increasing trend of Arg1- and iNOS-expressing microglia was observed in the cervical spinal cords of SOD1(G93A) mice. Additionally, Arg1-negative motor neurons appeared to selectively decline in the spinal cord of SOD1(G93A) mice, suggesting that Arg1 may have a neuroprotective function. CONCLUSIONS: This study suggests that the increase in spinal cord microglia occurs around and after disease onset and is preceded by cellular pathology. The results show that Arg1 and iNOS, thought to have opposing inflammatory properties, are upregulated in microglia during disease progression and that Arg1 in motor neurons may confer protection from disease processes. Further understanding of the neuroinflammatory response, and the Arg1/iNOS balance in motor neurons, may provide suitable therapeutic targets for ALS.

Congenital anomalies identified at birth among infants born following assisted reproductive technology in Colorado.

BACKGROUND: Assisted reproductive technology (ART) has assisted many infertile couples in conceiving. Despite increasing use in the United States, the association between ART and congenital anomalies remains a highly contested subject. We conducted a study to examine the risk of congenital anomalies among infants conceived using ART. METHODS: A retrospective cohort study of 344,567 infants born in Colorado from 2007 to 2011 was conducted using data obtained from the Colorado Birth Certificate Database. The incidence of congenital anomalies identified at birth following conception with ART was assessed and compared with all naturally conceived infants born during the same time period. The odds ratio was calculated using multiple logistic regression after adjusting for multiple confounders. RESULTS: Of 2071 infants, 23 (1.11%) conceived using ART had a congenital anomaly identified at birth compared with 3826 (1.12%) of 342,496 infants conceived naturally. The adjusted odds ratio of a congenital anomaly among infants born following conception with ART was 1.01 (95% confidence interval, 0.67-1.52). The proportion of infants born following usage of ART in Colorado has not changed significantly (p = 0.20) from 2007 to 2011 with an overall proportion of 0.60% (range 0.52-0.64%), while the incidence of congenital anomalies has decreased significantly (p = 0.002) during the study years with an average of 1.12% (range, 0.92-1.25%). CONCLUSION: This study suggests that conception by means of ART is not associated with an increased risk of congenital abnormalities identified by birth certificate data in Colorado when compared with births following natural conception.

Why should nanoscience students be taught to be ethically competent?

During the education of scientists at the university level the students become more and more specialized. The specialization of the students is a consequence of the scientific research becoming specialized as well. In the interdisciplinary field of nanoscience the importance of specialization is also emphasized throughout the education. Being an interdisciplinary field of study the specialization in this area is not focused on scientific disciplines, but on the different branches of the research. Historically ethics has not been a priority in science education, however, in recent years the importance of such teachings has been highly recognize especially in medicine, biotechnology and engineering. The rapid development, the many new and unknown areas and the highly specialized focus of nanotechnology suggest the importance of having ethically competent researchers. In this article the importance of ethical competence in nanoscience research is argued for by an example of a dilemma that could occur in a research project. The dilemma is analyzed using two different ethical views, generating two different choices for action. It is seen that the dilemma can have more than one solution and that ethical competence can help in justifying the choice of solution in a specific situation. Furthermore it is suggested that a way to reach this competence is through education in ethics incorporated into the nanoscience education curriculum.

Dynamics and activity of an ammonia-oxidizing archaea bloom in South San Francisco Bay.

Transient or recurring blooms of ammonia-oxidizing archaea (AOA) have been reported in several estuarine and coastal environments, including recent observations of AOA blooms in South San Francisco Bay. Here, we measured nitrification rates, quantified AOA abundance, and analyzed both metagenomic and metatranscriptomic data to examine the dynamics and activity of nitrifying microorganisms over the course of an AOA bloom in South San Francisco Bay during the autumn of 2018 and seasonally throughout 2019. Nitrification rates were correlated with AOA abundance in quantitative polymerase chain reaction (PCR) data, and both increased several orders of magnitude between the autumn AOA bloom and spring and summer seasons. From bloom samples, we recovered an extremely abundant, high-quality Candidatus Nitrosomarinus catalina-like AOA metagenome-assembled genome that had high transcript abundance during the bloom and expressed >80% of genes in its genome. We also recovered a putative nitrite-oxidizing bacteria metagenome-assembled genome from within the Nitrospinaceae that was of much lower abundance and had lower transcript abundance than AOA. During the AOA bloom, we observed increased transcript abundance for nitrogen uptake and oxidative stress genes in non-nitrifier metagenome-assembled genomes. This study confirms AOA are not only abundant but also highly active during blooms oxidizing large amounts of ammonia to nitrite-a key intermediate in the microbial nitrogen cycle-and producing reactive compounds that may impact other members of the microbial community.

Diverse and unconventional methanogens, methanotrophs, and methylotrophs in metagenome-assembled genomes from subsurface sediments of the Slate River floodplain, Crested Butte, CO, USA.

We use metagenome-assembled genomes (MAGs) to understand single-carbon (C1) compound-cycling-particularly methane-cycling-microorganisms in montane riparian floodplain sediments. We generated 1,233 MAGs (>50% completeness and <10% contamination) from 50- to 150-cm depth below the sediment surface capturing the transition between oxic, unsaturated sediments and anoxic, saturated sediments in the Slate River (SR) floodplain (Crested Butte, CO, USA). We recovered genomes of putative methanogens, methanotrophs, and methylotrophs (n = 57). Methanogens, found only in deep, anoxic depths at SR, originate from three different clades (Methanoregulaceae, Methanotrichaceae, and Methanomassiliicoccales), each with a different methanogenesis pathway; putative methanotrophic MAGs originate from within the Archaea (Candidatus Methanoperedens) in anoxic depths and uncultured bacteria (Ca. Binatia) in oxic depths. Genomes for canonical aerobic methanotrophs were not recovered. Ca. Methanoperedens were exceptionally abundant (~1,400× coverage, >50% abundance in the MAG library) in one sample that also contained aceticlastic methanogens, indicating a potential C1/methane-cycling hotspot. Ca. Methylomirabilis MAGs from SR encode pathways for methylotrophy but do not harbor methane monooxygenase or nitrogen reduction genes. Comparative genomic analysis supports that one clade within the Ca. Methylomirabilis genus is not methanotrophic. The genetic potential for methylotrophy was widespread, with over 10% and 19% of SR MAGs encoding a methanol dehydrogenase or substrate-specific methyltransferase, respectively. MAGs from uncultured Thermoplasmata archaea in the Ca. Gimiplasmatales (UBA10834) contain pathways that may allow for anaerobic methylotrophic acetogenesis. Overall, MAGs from SR floodplain sediments reveal a potential for methane production and consumption in the system and a robust potential for methylotrophy.IMPORTANCEThe cycling of carbon by microorganisms in subsurface environments is of particular relevance in the face of global climate change. Riparian floodplain sediments contain high organic carbon that can be degraded into C1 compounds such as methane, methanol, and methylamines, the fate of which depends on the microbial metabolisms present as well as the hydrological conditions and availability of oxygen. In the present study, we generated over 1,000 MAGs from subsurface sediments from a montane river floodplain and recovered genomes for microorganisms that are capable of producing and consuming methane and other C1 compounds, highlighting a robust potential for C1 cycling in subsurface sediments both with and without oxygen. Archaea from the Ca. Methanoperedens genus were exceptionally abundant in one sample, indicating a potential C1/methane-cycling hotspot in the Slate River floodplain system.

New insights into complex formation by SARS-CoV-2 nsp10 and nsp14.

SARS-CoV-2 non-structural protein 10 (nsp10) is essential for the stimulation of enzymatic activities of nsp14 and nsp16, acting as both an activator and scaffolding protein. Nsp14 is a bifunctional enzyme with the N-terminus containing a 3'-5' exoribonuclease (ExoN) domain that allows the excision of nucleotide mismatches at the virus RNA 3'-end, and a C-terminal N7-methyltransferase (N7-MTase) domain. Nsp10 is required for stimulating both ExoN proofreading and the nsp16 2'-O-methyltransferase activities. This makes nsp10 a central player in both viral resistance to nucleoside-based drugs and the RNA cap methylation machinery that helps the virus evade innate immunity. We characterised the interactions between full-length nsp10 (139 residues), N- and C-termini truncated nsp10 (residues 10-133), and nsp10 with a C-terminal truncation (residues 1-133) with nsp14 using microscale thermophoresis, multi-detection SEC, and hydrogen-deuterium (H/D) exchange mass spectrometry. We describe the functional role of the C-terminal region of nsp10 for binding to nsp14 and show that full N- and C-termini of nsp10 are important for optimal binding. In addition, our H/D exchange experiments suggest an intermediary interaction of nsp10 with the N7-MTase domain of nsp14. In summary, our results suggest intermediary steps in the process of association or dissociation of the nsp10-nsp14 complex, involving contacts between the two proteins in regions not identifiable by X-ray crystallography alone.

Therapeutic drug monitoring of imatinib - how far are we in the leukemia setting?

INTRODUCTION: Tyrosine kinase inhibitors (TKIs) have revolutionized survival rates of chronic myeloid leukemia (CML) and Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia (ALL) and replaced hematopoietic stem cell transplantation (hSCT) as the key treatment option for these patients. More recently, the so-called Philadelphia chromosome-like (Ph-like) ALL has similarly benefitted from TKIs. However, many patients shift from the first generation TKI, imatinib, due to treatment-related toxicities or lack of treatment efficacy. A more personalized approach to TKI treatment could counteract these challenges and potentially be more cost-effective. Therapeutic drug monitoring (TDM) has led to higher response rates and less treatment-related toxicity in adult CML but is rarely used in ALL or in childhood CML. AREAS COVERED: This review summarizes different antileukemic treatment indications for TKIs with focus on imatinib and its pharmacokinetic/-dynamic properties as well as opportunities and pitfalls of TDM for imatinib treatment in relation to pharmacogenetics and co-medication for pediatric and adult Ph+/Ph-like leukemias. EXPERT OPINION: TDM of imatinib adds value to standard monitoring of ABL-class leukemia by uncovering non-adherence and potentially mitigating adverse effects. Clinically implementable pharmacokinetic/-dynamic models adjusted for relevant pharmacogenetics could improve individual dosing. Prospective trials of TDM-based treatments, including both children and adults, are needed.

Hyper-recombination in ribosomal DNA is driven by long-range resection-independent RAD51 accumulation.

Ribosomal DNA (rDNA) encodes the ribosomal RNA genes and represents an intrinsically unstable genomic region. However, the underlying mechanisms and implications for genome integrity remain elusive. Here, we use Bloom syndrome (BS), a rare genetic disease characterized by DNA repair defects and hyper-unstable rDNA, as a model to investigate the mechanisms leading to rDNA instability. We find that in Bloom helicase (BLM) proficient cells, the homologous recombination (HR) pathway in rDNA resembles that in nuclear chromatin; it is initiated by resection, replication protein A (RPA) loading and BRCA2-dependent RAD51 filament formation. However, BLM deficiency compromises RPA-loading and BRCA1/2 recruitment to rDNA, but not RAD51 accumulation. RAD51 accumulates at rDNA despite depletion of long-range resection nucleases and rDNA damage results in micronuclei when BLM is absent. In summary, our findings indicate that rDNA is permissive to RAD51 accumulation in the absence of BLM, leading to micronucleation and potentially global genomic instability.

Pelagic metagenome-assembled genomes from an estuarine salinity gradient in San Francisco Bay.

San Francisco Bay (SFB) is a large and highly human-impacted estuarine system. We produced 449 metagenome-assembled genomes from SFB waters, collected along the salinity gradient, providing a rich data set to compare the metabolic potential of microorganisms from different salinity zones within SFB and to other estuarine systems.

Production and cross-feeding of nitrite within Prochlorococcus populations.

Prochlorococcus is an abundant photosynthetic bacterium in the open ocean, where nitrogen (N) often limits phytoplankton growth. In the low-light-adapted LLI clade of Prochlorococcus, nearly all cells can assimilate nitrite (NO2-), with a subset capable of assimilating nitrate (NO3-). LLI cells are maximally abundant near the primary NO2- maximum layer, an oceanographic feature that may, in part, be due to incomplete assimilatory NO3- reduction and subsequent NO2- release by phytoplankton. We hypothesized that some Prochlorococcus exhibit incomplete assimilatory NO3- reduction and examined NO2- accumulation in cultures of three Prochlorococcus strains (MIT0915, MIT0917, and SB) and two Synechococcus strains (WH8102 and WH7803). Only MIT0917 and SB accumulated external NO2- during growth on NO3-. Approximately 20-30% of the NO3- transported into the cell by MIT0917 was released as NO2-, with the rest assimilated into biomass. We further observed that co-cultures using NO3- as the sole N source could be established for MIT0917 and Prochlorococcus strain MIT1214 that can assimilate NO2- but not NO3-. In these co-cultures, the NO2- released by MIT0917 is efficiently consumed by its partner strain, MIT1214. Our findings highlight the potential for emergent metabolic partnerships that are mediated by the production and consumption of N cycle intermediates within Prochlorococcus populations. IMPORTANCE Earth's biogeochemical cycles are substantially driven by microorganisms and their interactions. Given that N often limits marine photosynthesis, we investigated the potential for N cross-feeding within populations of Prochlorococcus, the numerically dominant photosynthetic cell in the subtropical open ocean. In laboratory cultures, some Prochlorococcus cells release extracellular NO2- during growth on NO3-. In the wild, Prochlorococcus populations are composed of multiple functional types, including those that cannot use NO3- but can still assimilate NO2-. We show that metabolic dependencies arise when Prochlorococcus strains with complementary NO2- production and consumption phenotypes are grown together on NO3-. These findings demonstrate the potential for emergent metabolic partnerships, possibly modulating ocean nutrient gradients, that are mediated by cross-feeding of N cycle intermediates.

Recombinant Protein Production Using the Baculovirus Expression Vector System (BEVS).

The baculovirus expression vector system (BEVS) is one of the most popular eukaryotic systems for recombinant protein production. The focus of our protein production platform is the provision of recombinant proteins for research use, where generally only small quantities are required, in the range of tens of micrograms to a few hundred milligrams. Here, we present methods that reflect our standard operating procedures and setup to be able to frequently, and often repeatedly, produce many different types of proteins.

The potential effect of walking on quantitative sensory testing, pain catastrophizing, and perceived stress: an exploratory study.

OBJECTIVES: Studies suggest that a range of pain mechanisms, such as poor quality of sleep, perceived stress, pain catastrophizing or pain sensitivity, are likely to enhance clinical pain. Animal studies suggest that these pain mechanisms can be modulated by increasing physical activity, but human data are needed to support this hypothesis. This exploratory study aimed to investigate the changes in pain mechanisms after a simple self-directed walking program of 8-weeks. Additionally, this exploratory study investigated the interaction between changes over time in assessments of poor quality of sleep, perceived stress, pain catastrophizing or pain sensitivity and how these changes interacted with each other. METHODS: This prospective cohort study included 30 healthy subjects who were assessed at baseline and 4- and 8-weeks after initiating the walking program (30 min walking/day for 8 weeks). Self-report outcomes included: Pain Catastrophizing Scale (PCS), the Perceived Stress Scale (PSS) and Pittsburgh Sleep Quality Index. Pressure pain thresholds, temporal summation of pain and conditioned pain modulation (CPM) were assessed using cuff algometry. RESULTS: Twenty-four subjects completed all the visits (age: 42.2, SD: 14.9, 16 females). PCS and PSS significantly decreased at the 8-week's visit compared to baseline (p<0.05). No significant differences were seen for an improvement in quality of sleep (p=0.071) and pain sensitivity (p>0.075) when comparing the 8-week's visit to the baseline visit. Changes in pain mechanisms comparing baseline and 8-weeks data were calculated and regression analyses found that an improvement in PCS was associated with an improvement in CPM (R2=0.197, p=0.017) and that a higher adherence to the walking program was associated with a larger improvement in PCS (R2=0.216, p=0.013). CONCLUSIONS: The current exploratory study indicates that a simple self-directed walking program of 8-weeks can improve pain catastrophizing thoughts, perceived stress. Higher adherence to the walking program were associated with an improvement in pain catastrophizing and an improvement in pain catastrophizing was associated with an increase in conditioned pain modulation.