Forecasting freshwater cyanobacterial harmful algal blooms for Sentinel-3 satellite resolved U.S. lakes and reservoirs.

This forecasting approach may be useful for water managers and associated public health managers to predict near-term future high-risk cyanobacterial harmful algal blooms (cyanoHAB) occurrence. Freshwater cyanoHABs may grow to excessive concentrations and cause human, animal, and environmental health concerns in lakes and reservoirs. Knowledge of the timing and location of cyanoHAB events is important for water quality management of recreational and drinking water systems. No quantitative tool exists to forecast cyanoHABs across broad geographic scales and at regular intervals. Publicly available satellite monitoring has proven effective in detecting cyanobacteria biomass near-real time within the United States. Weekly cyanobacteria abundance was quantified from the Ocean and Land Colour Instrument (OLCI) onboard the Sentinel-3 satellite as the response variable. An Integrated Nested Laplace Approximation (INLA) hierarchical Bayesian spatiotemporal model was applied to forecast World Health Organization (WHO) recreation Alert Level 1 exceedance >12 µg L-1 chlorophyll-a with cyanobacteria dominance for 2192 satellite resolved lakes in the United States across nine climate zones. The INLA model was compared against support vector classifier and random forest machine learning models; and Dense Neural Network, Long Short-Term Memory (LSTM), Recurrent Neural Network (RNN), and Gneural Network (GNU) neural network models. Predictors were limited to data sources relevant to cyanobacterial growth, readily available on a weekly basis, and at the national scale for operational forecasting. Relevant predictors included water surface temperature, precipitation, and lake geomorphology. Overall, the INLA model outperformed the machine learning and neural network models with prediction accuracy of 90% with 88% sensitivity, 91% specificity, and 49% precision as demonstrated by training the model with data from 2017 through 2020 and independently assessing predictions with data from the 2021 calendar year. The probability of true positive responses was greater than false positive responses and the probability of true negative responses was less than false negative responses. This indicated the model correctly assigned lower probabilities of events when they didn't exceed the WHO Alert Level 1 threshold and assigned higher probabilities when events did exceed the threshold. The INLA model was robust to missing data and unbalanced sampling between waterbodies.

Assessing potential of the Geostationary Littoral Imaging and Monitoring Radiometer (GLIMR) for water quality monitoring across the coastal United States.

The Geostationary Littoral Imaging and Monitoring Radiometer (GLIMR) will provide unique high temporal frequency observations of the United States coastal waters to quantify processes that vary on short temporal and spatial scales. The frequency and coverage of observations from geostationary orbit will improve quantification and reduce uncertainty in tracking water quality events such as harmful algal blooms and oil spills. This study looks at the potential for GLIMR to complement existing satellite platforms from its unique geostationary viewpoint for water quality and oil spill monitoring with a focus on temporal and spatial resolution aspects. Water quality measures derived from satellite imagery, such as harmful algal blooms, thick oil, and oil emulsions are observable with glint <0.005 sr-1, while oil films require glint >10-5 sr-1. Daily imaging hours range from 6 to 12 h for water quality measures, and 0 to 6 h for oil film applications throughout the year as defined by sun glint strength. Spatial pixel resolution is 300 m at nadir and median pixel resolution was 391 m across the entire field of regard, with higher spatial resolution across all spectral bands in the Gulf of Mexico than existing satellites, such as MODIS and VIIRS, used for oil spill surveillance reports. The potential for beneficial glint use in oil film detection and quality flagging for other water quality parameters was greatest at lower latitudes and changed location throughout the day from the West and East Coasts of the United States. GLIMR scan times can change from the planned ocean color default of 0.763 s depending on the signal-to-noise ratio application requirement and can match existing and future satellite mission regions of interest to leverage multi-mission observations.

Satellite and in situ cyanobacteria monitoring: Understanding the impact of monitoring frequency on management decisions.

Cyanobacterial harmful algal blooms (cyanoHABs) in reservoirs can be transported to downstream waters via scheduled discharges. Transport dynamics are difficult to capture in traditional cyanoHAB monitoring, which can be spatially disparate and temporally discontinuous. The introduction of satellite remote sensing for cyanoHAB monitoring provides opportunities to detect where cyanoHABs occur in relation to reservoir release locations, like canal inlets. The study objectives were to assess (1) differences in reservoir cyanoHAB frequencies as determined by in situ and remotely sensed data and (2) the feasibility of using satellite imagery to identify conditions associated with release-driven cyanoHAB export. As a representative case, Lake Okeechobee and the St. Lucie Estuary (Florida, USA), which receives controlled releases from Lake Okeechobee, were examined. Both systems are impacted by cyanoHABs, and the St. Lucie Estuary experienced states of emergency for extreme cyanoHABs in 2016 and 2018. Using the European Space Agency's Sentinel-3 OLCI imagery processed with the Cyanobacteria Index (CIcyano), cyanoHAB frequencies across Lake Okeechobee from May 2016-April 2021 were compared to frequencies from in situ data. Strong agreement was observed in frequency rankings between the in situ and remotely sensed data in capturing intra-annual variability in bloom frequencies across Lake Okeechobee (Kendall's tau = 0.85, p-value = 0.0002), whereas no alignment was observed when evaluating inter-annual variation (Kendall's tau = 0, p-value = 1). Further, remotely sensed observations revealed that cyanoHABs were highly frequent near the inlet to the canal connecting Lake Okeechobee to the St. Lucie Estuary in state-of-emergency years, a pattern not evident from in situ data alone. This study demonstrates how remote sensing can complement traditional cyanoHAB monitoring to inform reservoir release decision making.

Systematic microRNA analysis identifies ATP6V0C as an essential host factor for human cytomegalovirus replication.

Recent advances in microRNA target identification have greatly increased the number of putative targets of viral microRNAs. However, it is still unclear whether all targets identified are biologically relevant. Here, we use a combined approach of RISC immunoprecipitation and focused siRNA screening to identify targets of HCMV encoded human cytomegalovirus that play an important role in the biology of the virus. Using both a laboratory and clinical strain of human cytomegalovirus, we identify over 200 putative targets of human cytomegalovirus microRNAs following infection of fibroblast cells. By comparing RISC-IP profiles of miRNA knockout viruses, we have resolved specific interactions between human cytomegalovirus miRNAs and the top candidate target transcripts and validated regulation by western blot analysis and luciferase assay. Crucially we demonstrate that miRNA target genes play important roles in the biology of human cytomegalovirus as siRNA knockdown results in marked effects on virus replication. The most striking phenotype followed knockdown of the top target ATP6V0C, which is required for endosomal acidification. siRNA knockdown of ATP6V0C resulted in almost complete loss of infectious virus production, suggesting that an HCMV microRNA targets a crucial cellular factor required for virus replication. This study greatly increases the number of identified targets of human cytomegalovirus microRNAs and demonstrates the effective use of combined miRNA target identification and focused siRNA screening for identifying novel host virus interactions.

Peptide fragments of a beta-defensin derivative with potent bactericidal activity.

Beta-defensins are known to be both antimicrobial and able to chemoattract various immune cells. Although the sequences of paralogous genes are not highly conserved, the core defensin structure is retained. Defb14-1C(V) has bactericidal activity similar to that of its parent peptide (murine beta-defensin Defb14) despite all but one of the canonical six cysteines being replaced with alanines. The 23-amino-acid N-terminal half of Defb14-1C(V) is a potent antimicrobial while the C-terminal half is not. Here, we use a library of peptide derivatives to demonstrate that the antimicrobial activity can be localized to a particular region. Overlapping fragments of the N-terminal region were tested for their ability to kill Gram-positive and Gram-negative bacteria. We demonstrate that the most N-terminal fragments (amino acids 1 to 10 and 6 to 17) are potent antimicrobials against Gram-negative bacteria whereas fragments based on sequence more C terminal than amino acid 13 have very poor activity against both Gram-positive and -negative types. We further test a series of N-terminal deletion peptides in both their monomeric and dimeric forms. We find that bactericidal activity is lost against both Gram types as the deletion region increases, with the point at which this occurs varying between bacterial strains. The dimeric form of the peptides is more resistant to the peptide deletions, but this is not due just to increased charge. Our results indicate that the primary sequence, together with structure, is essential in the bactericidal action of this beta-defensin derivative peptide and importantly identifies a short fragment from the peptide that is a potent bactericide.

Isoleucine/leucine2 is essential for chemoattractant activity of beta-defensin Defb14 through chemokine receptor 6.

Beta-defensins are both antimicrobial and able to chemoattract various immune cells including immature dendritic cells and CD4 T cells through CCR6. They are short, cationic peptides with a highly conserved six-cysteine motif. It has been shown that only the fifth cysteine is critical for chemoattraction of cells expressing CCR6. In order to identify other residues essential for functional interaction with CCR6 we used a library of peptide deletion derivatives based on Defb14. Loss of the initial two amino acids from the Defb14-1C(V) derivative destroys its ability to chemoattract cells expressing CCR6. As the second amino acid is an evolutionarily conserved leucine, we make full-length Defb14-1C(V) peptides with substitution of the leucine(2) for glycine (L2G), lysine (L2K) or isoleucine (L2I). Defb14-1C(V) L2G and L2K and are unable to chemoattract CCR6 expressing cells but the semi-conservative change L2I has activity. By circular dichroism spectroscopy we can see no evidence for a significant change in secondary structure as a consequence of these substitutions and so cannot attribute loss of chemotactic activity with disruption of the N-terminal helix. We conclude that isoleucine/leucine in the N-terminal alpha-helix region of this beta-defensin is essential for CCR6-mediated chemotaxis.

Peptide thioester synthesis through N-->S acyl-transfer: application to the synthesis of a beta-defensin.

Peptide thioesters readily prepared through N-->S acyl transfer of a specific C-terminal motif provide access to biologically active mini-proteins using native chemical ligation.

Defensin-related peptide 1 (Defr1) is allelic to Defb8 and chemoattracts immature DC and CD4+ T cells independently of CCR6.

Beta-defensins comprise a family of cationic, antimicrobial and chemoattractant peptides. The six cysteine canonical motif is retained throughout evolution and the disulphide connectivities stabilise the conserved monomer structure. A murine beta-defensin gene (Defr1) present in the main defensin cluster of C57B1/6 mice, encodes a peptide with only five of the canonical six cysteine residues. In other inbred strains of mice, the allele encodes Defb8, which has the six cysteine motif. We show here that in common with six cysteine beta-defensins, defensin-related peptide 1 (Defr1) displays chemoattractant activity for CD4(+) T cells and immature DC (iDC), but not mature DC cells or neutrophils. Murine Defb2 replicates this pattern of attraction. Defb8 is also able to attract iDC but not mature DC. Synthetic analogues of Defr1 with the six cysteines restored (Defr1 Y5C) or with only a single cysteine (Defr1-1c(V)) chemoattract CD4(+) T cells with reduced activity, but do not chemoattract DC. Beta-defensins have previously been shown to attract iDC through CC receptor 6 (CCR6) but neither Defr1 or its related peptides nor Defb8, chemoattract cells overexpressing CCR6. Thus, we demonstrate that the canonical six cysteines of beta-defensins are not required for the chemoattractant activity of Defr1 and that neither Defr1 nor the six cysteine polymorphic variant allele Defb8, act through CCR6.

mc1r Pathway regulation of zebrafish melanosome dispersion.

Zebrafish rapidly alter their pigmentation in response to environmental changes. For black melanocytes, this change is due to aggregation or dispersion of melanin in the cell. Dispersion and aggregation are controlled by intracellular cyclic adenosine monophosphate (cAMP) levels, which increase upon stimulation by alpha melanocyte-stimulating hormone (alpha-MSH) or reduce with melanin-concentrating hormone (MCH). In mammals and birds, the melanocortin-1-receptor (MC1R) responds to MSH, and stimulates the synthesis of black eumelanin. While MSH-cAMP signaling stimulates melanogenesis in mammals, and melanosome dispersal in cold-blood vertebrates, the pathway components are highly conserved. However, it has only been assumed that mc1r mediates melanosome dispersal in fish. Here, using morpholino oligonucleotides designed to knockdown mc1r expression, we find that mc1r morphants are unable to disperse melanosomes when grown in dark conditions. We also use chemical modifiers of the cAMP pathway, and find an unexpected response to the specific phosphodiesterase 4 (PDE4) inhibitor, rolipram, in melanosome dispersal. When treated with the drug, melanosomes fail to fully disperse in dark conditions, despite presumed increased levels of cAMP, and in contrast to the effects of the nonselective PDE inhibitor, 3-isobutyl-1-methylxanthine. In conclusion, we demonstrate a direct role for mc1r in zebrafish melanosome dispersal in response to background, and use chemical modification of this pathway to uncover a possible new layer of regulation in melanosome dispersal in zebrafish.

Microcephalin coordinates mitosis in the syncytial Drosophila embryo.

Microcephalin (MCPH1) is mutated in primary microcephaly, an autosomal recessive human disorder of reduced brain size. It encodes a protein with three BRCT domains that has established roles in DNA damage signalling and the cell cycle, regulating chromosome condensation. Significant adaptive evolutionary changes in primate MCPH1 sequence suggest that changes in this gene could have contributed to the evolution of the human brain. To understand the developmental role of microcephalin we have studied its function in Drosophila. We report here that Drosophila MCPH1 is cyclically localised during the cell cycle, co-localising with DNA during interphase, but not with mitotic chromosomes. mcph1 mutant flies have a maternal effect lethal phenotype, due to mitotic arrest occurring in early syncytial cell cycles. Mitotic entry is slowed from the very first mitosis in such embryos, with prolonged prophase and metaphase stages; and frequent premature separation as well as detachment of centrosomes. As a consequence, centrosome and nuclear cycles become uncoordinated, resulting in arrested embryonic development. Phenotypic similarities with abnormal spindle (asp) and centrosomin (cnn) mutants (whose human orthologues are also mutated in primary microcephaly), suggest that further studies in the Drosophila embryo may establish a common developmental and cellular pathway underlying the human primary microcephaly phenotype.

Implementation and case-study results of potentially better practices to improve pain management of neonates.

OBJECTIVE: Collaborative quality improvement techniques were used to facilitate local quality improvement in the management of pain in infants. Several case studies are presented to highlight this process. METHODS: Twelve NICUs in the Neonatal Intensive Care Quality Improvement Collaborative 2002 focused on improving neonatal pain management and sedation practices. These centers developed and implemented evidence-based potentially better practices for pain management and sedation in neonates. The group introduced changes through plan-do-study-act cycles and tracked performance measures throughout the process. RESULTS: Strategies for implementing potentially better practices varied between centers on the basis of local characteristics. Individual centers identified barriers to implementation, developed tools for improvement, and shared their experience with the collaborative. Baseline data from the 12 sites revealed substantial opportunities for improved pain management, and local potentially better practice implementation resulted in measurable improvements in pain management at participating centers. CONCLUSIONS: The use of collaborative quality improvement techniques enhanced local quality improvement efforts and resulted in effective implementation of potentially better practices at participating centers.