microRNAs (miRNAs) are one kind of small non-coding RNAs widely distributed in insects. Many studies have shown that miRNAs play critical roles in development, differentiation, apoptosis, and innate immunity. However, there are a few reports describing miRNAs in Anopheles sinensis, the most common, and one of the dominant malaria mosquito in China. Here, we investigated the global miRNA expression profile across four different developmental stages including embryo, larval, pupal, and adult stages using Illumina Hiseq 2500 sequencing.
In total, 164 miRNAs were obtained out of 107.46 million raw sequencing reads. 99 of them identified as known miRNAs, and the remaining 65 miRNAs were considered as novel. By analyzing the read counts of miRNAs in all developmental stages, 95 miRNAs showed stage-specific expression (q < 0.01 and |log2 (fold change)| > 1) in consecutive stages, indicating that these miRNAs may be involved in critical physiological activity during development. Sixteen miRNAs were identified to be commonly dysregulated throughout four developmental stages. Many miRNAs showed stage-specific expression, such as asi-miR-2943 was exclusively expressed in the embryo stage, and asi-miR-1891 could not be detected in larval stage. The expression of six selected differentially expressed miRNAs identified by qRT-PCR were consistent with our sequencing results. Furthermore, 5296 and 1902 target genes were identified for the dysregulated 68 known and 27 novel miRNAs respectively by combining miRanda and RNAhybrid prediction. GO annotation and KEGG pathway analysis for the predicted genes of dysregulated miRNAs revealed that they might be involved in a broad range of biological processes related with the development, such as membrane, organic substance transport and several key pathways including protein processing in endoplasmic reticulum, propanoate metabolism and folate biosynthesis. Thirty-two key miRNAs were identified by microRNA-gene network analysis.
The present study represents the first global characterization of An. sinensis miRNAs in its four developmental stages. The presence and differential expression of An. sinensis miRNAs imply that such miRNAs may play critical roles in An. sinensis life cycle. A better understanding of the functions of these miRNAs will have great implication for the effective control of vector population and therefore interrupting malaria transmission.
Anopheles sinensis is one of the most important disease vectors in China. It is considered as the primary vector of P. vivax malaria due to its wide distribution and high density in central China where several malaria outbreaks occurred in history [1, 2]. Like many mosquitoes, An. sinensis goes through four life stages that include egg, larva, pupa and the adult. During these developmental periods, a fine-tuning complex biological process, such as embryogenesis, organ differentiation, and metamorphosis, etc., is completed.
For the past decades, facilitated by high-throughput sequencing technology, a large number of small non-coding RNAs (ncRNAs) have been discovered in many species [3, 4]. Amongst them, microRNAs (miRNAs) have become the most popular research topic for its critical role in regulating important biological events at the post-transcriptional level. Mature miRNAs are single-stranded, evolutionarily conserved endogenous small ncRNAs of approximately 22 nucleotides (nt) in length, which could lead to transcriptional decay through translational inhibition, transcript degradation or both [5, 6].
The first miRNA repertoire of mosquito species was reported from the African malaria vector Anopheles gambiae . Since then, mosquito miRNAs have been identified and characterized in a number of mosquito species, including Anopheles stephensi [8, 9], Aedes aegypti [10–12], Culex fatigans [13–15], and many others, but not yet in An. sinensis. Several studies suggested that miRNAs may contribute to an important regulatory role in the post-transcriptional level in developmental stages. For instance, temporal and stage specific expression of miRNAs has been identified in the embryo, larval, pupal and adult stage of Ae. albopictus , An. anthropophagus , and An. stephensi . Among them, the expression of aal-miR-286b was increased in the embryo, while aal-miR-2942 had the highest level of expression in larvae although it was normally expressed at the embryo, pupal and adult stages . In addition, ast-miR-1891, ast-miR-190-3p, ast-miR-285, ast-miR-988-3p, and ast-miR-989 were absent in the larval stage, while ast-miR-8-3p was the most abundant in both male and female larval stage . These results indicated that dysregulated miRNAs might play pivotal roles in embryogenesis and metamorphosis throughout development. However, the exact functions of these miRNAs remain to be determined.
In this study, we provided a comprehensive repertoire of An. sinensis miRNAs and investigated the differential expression of miRNAs using Solexa sequencing technology, with an aim to identify key regulatory miRNAs through development stages. Selected candidate miRNAs were then validated by qRT-PCR in samples that have been used in sequencing. To explore the potential gene regulatory networks, 32 dysregulated miRNAs were defined as key miRNAs during development and then subjected to miRNA-gene network analysis in terms of developmental pathway analysis. The data described here served as an important step towards the understanding of gene expression modulation by miRNAs in An. sinensis, which will further advance functional studies for novel mosquito control approaches developing and promote elimination campaign in China.
Anopheles sinensis (China strain) from insectary were reared at 28 ± 2 °C, 70–75% humidity in the Vector Control Reference Laboratory at the National Institute of Parasitic Diseases (NIPD), Shanghai, China. Adult mosquitoes were maintained in screened cages, and provided constant access to water and glucose-soaked sponges.
Sample collections from different developmental stages of An. sinensis for high throughput sequencing are briefly described below. Embryos were collected within 24 h after placing a damp collection filter paper within the cage and were pooled to represent the embryonic stage. In the larval sample collection, we used mixed larval sample (I–IV instars) including early and late larval samples. Pupal samples were collected from a pool of varied ages. Female adults were collected after emerging one to 3 days. All samples were flash frozen in liquid nitrogen immediately following collection, and then stored at − 80 °C until for RNA isolation.
Briefly, total RNAs were extracted separately from the four different life cycle stages of An. sinensis (eggs, larvae, pupae and unfed adult females) using TRIzol reagent (Invitrogen, USA) according to the manufacturer's protocol. RNA purity was checked using the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA). RNA degradation and contamination were monitored on 1% agarose gels. RNA concentration was measured using Qubit® RNA Assay Kit in Qubit® 2.0 Fluorometer (Life Technologies, CA, USA). RNA quality assessment was determined by using Agilent 2100 Bioanalyzer RNA Nano 6000 kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). No rRNA depletion was performed. RNA extracts of the four life stages were sent to Newgenes Inc (Shanghai, China) for small RNA sequencing. A total amount of 3 µg of total RNA was ligated with 3′ and 5′ adaptors followed by reverse transcription using RT primers. The libraries were size-selected for sequencing of RNA fragments of 18–30 nucleotides. These small RNA libraries were then sequenced using Illumina Hiseq 2500 platform (Illumina Inc., USA).
Following sequencing, raw data (raw reads) of fastq format were firstly processed through custom perl and python scripts. In this step, clean data (clean reads) were obtained by removing reads containing ploy-N, with 5′ adapter contaminants, without 3′ adapter or the insert tag, containing ploy A or T or G or C and low quality reads from raw data. At the same time, Q20, Q30, and GC-content of the raw data were calculated. Then, a certain range of clean reads from each stage was combined to do all the downstream analyses. All reads were mapped to the An. sinensis strain China genome [GenBank: GCA_000441895.2] by Bowtie  without mismatch to analyze their expression and distribution on the reference.
For identification of known and novel miRNA sequences present in the four life stage datasets. Firstly, after removing tags originating from protein-coding genes, repeat sequences, rRNA, tRNA, snRNA, and snoRNA, small RNA tags were mapped to RepeatMasker, Rfam database or those types of data from Anopheles gambiae. Mapped small RNA tags were used to look for known miRNA using miRBase21  as reference. Custom scripts adapted from quantitative script of miRDeep2 were used to obtain the miRNA counts as well as base bias on the first position of identified miRNAs with certain length and on each position of all identified miRNAs. The reads that did not yield a match were used to predict novel miRNAs. The available software miREvo  and mirdeep2  were integrated to predict novel miRNA from the small RNA tags unannotated in the former steps. A number of stringent criterions were used for evaluating whether novel miRNA candidates were genuine miRNAs, such as exploring the secondary structure by RNAfold, calculating minimum free energy (MFE) and avoiding isolated base pairs. In each library, same sequences were pooled together to generate expression files (fasta) with their read count and unique ID used for further analysis.
All identified known miRNAs were named according to their most similar miRNAs in the miRNA database (miRBase) release 21 (June 2017). All identified novel miRNAs were named sequentially following the asi-mir-nov1. Raw sequencing data were prepared to submit to the National Center for Biotechnology Information (NCBI).
In order to analyze the relative abundance and expression level of miRNAs, tags per million of total RNA reads (TPM) for each miRNA in all four libraries were calculated. TPM was compared between the libraries to identify miRNAs differentially expressed between two consecutive life stages in the mosquito. Differential expression analysis of two samples was performed using the DEGseq  R package. p value was adjusted using q value . q < 0.01 and |log2 (fold change)| > 1 was set as the threshold for significantly differential expression by default.
To predict the potential targeted genes of miRNA, the targeted mRNAs of differentially expressed known and novel miRNAs were predicted by combining miRanda  and RNAhybrid  to select and assess overlapping predicted targets between each program. Briefly, 3′UTR of An. gambiae downloaded from vectorbase were used for the prediction. The targets showing complementarity with miRNA seed region and binding energy ≤ 20 kcal/mol were selected. The predicted targets were used for further GO and KEGG enrichment analysis.
Gene ontology (GO) enrichment analysis was used on the target gene candidates of differentially expressed miRNAs ("target gene candidates" in the following). GOseq based Wallenius non-central hyper-geometric distribution , which could adjust for gene length bias, was implemented for GO enrichment analysis. GO terms with corrected p value less than 0.05 were considered significantly enriched by target genes. KEGG pathway analyses were performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) (http://www.genome.ad.jp/kegg/) database . In this study, we used KOBAS  software to test the statistical enrichment of the target gene candidates in KEGG pathways, with the threshold of corrected p < 0.05.
To identify the key miRNAs in An. sinensis development, microRNA-gene network analysis was conducted for the commonly dysregulated miRNAs identified from the microRNA expression files, the exclusively expressed miRNAs in certain stages (TPM > 10) and the most significantly over/under-expressed miRNAs (>tenfold) and their predicted target developmental genes. Developmental genes of An. gambiae were accessed based on gene annotation in VectorBase  and through intensive literature surveys [30–33]. Interactive networks between key miRNAs and their target developmental genes were constructed by using Cytoscape .
To validate the presence and expression level of the identified miRNAs, nine miRNAs with differential expression were selected for qRT-PCR as described previously . The total RNA was extracted using TRIzol Reagent (Invitrogen, USA). The reverse transcription reaction was performed with RevertAid First Strand cDNA Synthesis Kit (Fermentas, USA), and the reverse-transcribed products were used as the template for qRT-PCR with miRNA-specific primers (Additional file 1: Table S1). All reactions were assayed in three biological and technical replications, and performed in an ABI-7300 (Applied Biosystems, USA) using SYBR Green qPCR kit (Thermo, USA). Quantitative measurements were normalized to the internal control of U6 small nuclear (U6), and relative expression was calculated by using the 2−ΔΔCt method .
We performed small RNA sequencing from eggs, larva, pupa and adult mosquito batches to identify An. sinensis miRNAs expressed during developmental stages. All sequencing data can be found in NCBISRA (PRJNA412443). In total, sequencing of four libraries yielded about 10.75 × 107 reads. After trimming the adaptors, 1.99 × 107 (96.71%), 2.85 × 107 (92.81%), 2.91 × 107 (86.06%) and 2.06 × 107 (92.38%) clean reads from ASE (egg), ASL (larva), ASP (pupa) and ASA (adult) libraries respectively were utilized for further processing (Table 1). The total number of the reads mapped to An. sinensis genome constitutes 82.53, 77.61, 80.55 and 85.75% of the total high-quality sRNA (small RNA) reads from eggs, larvae, pupae, and adult libraries, respectively (Table 1).
Among the libraries, size distribution of reads varied between 18 and 35 nt. We found that one peak with 21 nt in all libraries, followed by another peak with 20–23 nt which represents miRNAs (Fig. 1). This was consistent with the typical size of miRNAs in other reports. There was also a peak at 24–27 nt in almost all developmental libraries, which corresponds to piRNA-like small RNAs suggesting a possible role in embryogenesis and maintaining genome stability during development.
In order to annotate other small non-coding RNAs in the four libraries, all clean reads were aligned against Rfam version 11 . As showed in Fig. 2, the most abundant class of small non-coding RNAs in the eggs' library was miRNAs (50.10%). However, rRNAs were most abundant in the larvae (6.61%) and pupae library (5.8%), and tRNAs in the adult females' library (6.87%).
Based on previously known mosquito mature miRNAs in miRBase release 21 (June 2017), we discovered 99 known miRNAs in An. sinensis (Table 2) throughout four development stages. Reads that do not match any known miRNA sequences were further employed by miREvo and mirdeep2 to investigate the folding properties of precursors or hairpins for novel miRNAs. Finally, 65 mature miRNAs were identified as novel (default parameters of miREvo and mirdeep2) (Additional file 2: Table S2). Thus, a total of 164 An. sinensis miRNAs were identified in four life stages libraries of An. sinensis.
The expression patterns of miRNAs in different life stages were examined based on the number of reads revised. The result showed that the expression profiles of miRNAs varied from highly specific to ubiquitous during the four stages. One-hundred and seventeen miRNAs were ubiquitously expressed in all developmental stages, while some miRNAs showed stage-specific patterns (Fig. 3). Among the known miRNAs, asi-miR-276-3p, asi-miR-1, asi-miR-100, asi-miR-8-3p, asi-miR-999, and asi-miR-9a showed constant high expression throughout four development stages. asi-miR-276-3p was the most frequently expressed miRNA in the larvae, the pupae, and the adult females' library (TPM > 3.17 × 105), whereas asi-miR-1 was the dominant miRNA in the eggs' library (TPM > 1.87 × 105). Nevertheless, asi-miR-219 had the lowest expression level in all developmental stages. asi-miR-2943 was specifically expressed in the embryo stage, and asi-miR-1891 could not be detected in the larval stage. The highly expressed novel miRNA in the eggs and pupae libraries was asi-miR-nov54 (TPM = 0.45 × 103 and TPM = 1.50 × 103), while in the larvae library and adult females' library, it was asi-miR-nov55 (TPM = 0.91 × 103) and asi-miR-nov1 (TPM = 0.42 × 103), respectively. However, the expression level of novel miRNAs was generally low throughout all developmental stages when compared with known miRNAs.
Hierarchical heat map revealed different expression patterns of the miRNAs between the two consecutive developmental stages (Fig. 3). Based on q-value and log fold change, 33 significantly down-regulated miRNAs (26 for known and 7 for novel miRNAs) and 41 significantly up-regulated miRNAs (25 for known and 16 for novel) were identified from the embyro to the larval stage. 20 down-regulated miRNAs (18 for known and 2 for novel miRNAs) and 22 up-regulated miRNAs (18 for known and 4 for novel) were observed form the larval to the pupal stage. 22 significantly down-regulated miRNAs (14 for known and 8 for novel miRNAs) and 15 significantly up-regulated miRNAs (12 for known and 3 for novel) were expressed form the pupal to adult stage (Fig. 4). It is noteworthy that the total number of dysregulated miRNAs decreased from the embryo to the adult stage. We also identified two known miRNAs (asi-miR-2943 and asi-miR-286b) to be significantly over-expressed (>tenfold) form the embryo to the larval stage and one known miRNA (asi-miR-1891) to be significantly under-expressed form the pupal to the adult stage (>tenfold) (Fig. 4). Sixteen miRNAs were differentially expressed in all developmental stages of the mosquito. Based on the analysis of these regulated miRNAs between the two consecutive developmental stages, five miRNAs (asi-miR-1890, asi-miR-193, asi-miR-263a-5p, asi-miR-263b-5p and asi-miR-nov54) were down-regulated in the larval stage whereas get up-regulated in the pupal stage but down-regulated in the adult stage again. Seven miRNAs (asi-miR-1175-3p, asi-miR-1175-5p, asi-miR-281-3p, asi-miR-281-5p, asi-miR-317, asi-miR-34-5p and asi-miR-nov55) were up-regulated in the larval stage whereas get down-regulated in the pupal but up-regulated in the adult stage again. asi-miR-nov23 and asi-miR-2765 were up-regulated in the larval stage whereas get down-regulated in both pupal and adult stage. asi-miR-2944a-5p was down-regulated in the larval and the pupal stages but up-regulated in the adult stage. asi-miR-989 was down-regulated in larval and pupal stages but up-regulated in the adult stages.
To validate the small RNA sequencing results, we performed quantitative real-time PCR analysis by selecting nine miRNAs that showed dramatic changes in expression level at different developmental stages. The result (Fig. 5) showed that six miRNAs (asi-miR-276-3p, asi-miR-2943, asi-miR-1891, asi-miR-286b, asi-miR-1175-3p, and asi-miR-nov54) showed similar expression patterns as those revealed by our sequencing result. However, three miRNAs (asi-miR-263a-5p, asi-miR-nov23, and asi-miR-nov55) expression levels detected by qRT-PCR were inconsistent with that of our sequencing results due to some unknown reasons.
To understand the role of dysregulated miRNAs in physiological functions and biologic processes during development in An. sinensis, miRNA target gene prediction was performed to identify the putative target genes based on miRNA/mRNA complementarity. Multiple/different miRNA target prediction algorithms were applied to minimize the number of putative or false positive target genes. The target gene candidates of the differentially expressed miRNAs between two consecutive developmental stages of An. sinensis were predicted. A total of 7198 annotated mRNA transcripts were predicted as target gene candidates for 95 differentially expressed miRNAs (Additional file 3: Table S3). Among them, 5296 and 1902 target gene candidates were identified for 68 known and 27 novel miRNAs, respectively.
GO term annotation and KEGG Pathway analysis were used to further understand biological functions and signaling pathways that could potentially be regulated by these putative target genes. We found that the putative target genes of the differentially expressed miRNAs between two consecutive developmental stages of An. sinensis were involved in a broad range of biological functions and signaling pathways (Fig. 6). Detailed classifications about the biological process, cellular component, and molecular function of the miRNA targets were shown in Fig. 6a. By GO annotation, targets of miRNAs regulated from the embryo to the larval stage in the mosquito were enriched in intracellular, cytoplasm and cellular process. Targets of miRNAs regulated from larva to pupal stage in the mosquito were enriched in membrane part, membrane, cytoplasm and organic substance transport. Targets of miRNAs regulated from the pupa to the adult stage in the mosquito were enriched in structural molecule activity, membrane part and transport. Similarly, by KEGG pathway analysis, we found that 119 enriched pathways were identified in this study. The most enriched pathway included protein processing in endoplasmic reticulum, propanoate metabolism, folate biosynthesis, Citrate cycle (TCA cycle) and valine, leucine and isoleucine degradation (Fig. 6b).
Through analysis, 16 commonly dysregulated miRNAs identified from the miRNA expression files, 15 exclusively expressed miRNAs in certain stages (TPM > 10), and three the most significantly over/under-expressed miRNAs (>tenfold) were defined as the key miRNAs in An. sinensis development. A total of 546 developmental genes were obtained based on gene annotation in VectorBase and intensive literature surveys. After removing duplicates and invalid target genes, miRNA-gene network was built based on 32 key miRNAs and 104 developmental genes (Additional file 4: Table S4). We developed a network to demonstrate the overlapping miRNA targets for miRNAs since miRNAs usually have many mRNA targets. The number of targets for asi-miR-317, asi-miR-2944a-5p, asi-miR-263b-5p, asi-miR-263a-5p, and asi-miR-2943 was 10, 7, 6, 6 and 5, respectively (Fig. 7). This network also indicated that several mRNAs were predicted to be targets of one miRNAs. For example, AGAP001999, AGAP007696, AGAP005245, and AGAP004050 were targeted by two to five miRNAs, respectively (Fig. 7). As expected, the overall pattern suggests a positive correlation between the miRNA expression and developmental genes, such as AGAP000048 (APC), AGAP005449 (Cbl), AGAP001867 (hep), which encoding important components of major developmental signaling pathways.
In the present study, the miRNA expression profile of An. sinensis in four different life stages was analyzed by NGS technology, and approximately 100 million high-quality reads were obtained. The mapping rates of high-quality reads to the An. sinensis genome were between 77.61 and 85.75%. This might be subject to incomplete reference genome. The size distribution of small RNA sequence reads showed peaks between 20 AND 30 nucleotides comparing to the average miRNA length. Annotation of the sRNAs in the four libraries indicated that the most abundant class of sRNAs in the eggs, larvae, pupae and adult females' libraries was miRNAs, rRNAs (larvae and pupae) and tRNAs respectively. This result is consistent with previous research in Anopheles funestus , indicating that protein synthesis during development is of great importance.
In total, 99 known miRNAs and 65 novel miRNAs expressed in the four life stages of the An. sinensis were identified. As expected, most of the known miRNAs identified in An. sinensis were highly conserved across diverse mosquito species, suggesting that the conserved miRNAs may involve in many critically fundamental regulatory pathways. Novel miRNAs found in this study are not conserved in the Anopheles genus, and we cannot find homologs in other insect species by searching the miRBase, suggesting that these might be species-specific miRNAs. However, the predicted novel miRNAs exhibited much lower expression levels when compared with conserved miRNAs, which is also consistent with previous reports [9, 37]. Previously, Dritsou and his colleagues  computationally predicted 115 miRNAs (after removing 33 duplicates and 11 putatively new miRNA, 71 left) in An. sinensis from the genome level. We compared our miRNA dataset with theirs and found that 33 miRNAs overlap with our predictions. Among these miRNAs, 19 miRNAs could be found in Ae. aegypti, An. gambiae, and Cu. quinquefasciatus. Six miRNAs (miR-193, miR-2765, miR-277-5p miR-2b, miR-315-5p and miR-9b) were only found in Ae. aegypti. In contrast, we validate more miRNAs existence and detect their expression pattern at different developmental stages.
To obtain insight into possible stage-specific miRNAs and their roles in An. sinensis, we investigated the expression profile of the miRNAs identified in the four life stage libraries. One-hundred and seventeen miRNAs were ubiquitously expressed in all developmental stages. The top five most highly expressed miRNA across four developmental stages were asi-miR-276-3p, asi-miR-1, asi-miR-100, asi-miR-9a and miR-8, which is basically in line with previous report in other mosquito species [15, 38]. The most highly expressed miRNA in female An. sinensis was asi-miR-276-3p. In contrast, Skalsky et al.  reported that miR-184 was the most highly expressed miRNA in C7/10 Ae. albopictus cells and blood-fed female Cx. quinquefasciatus mosquitoes . afu-miR-8 was the dominant miRNA in the unfed adult An. funestus females . In addition, miR-1, miR-184, and miR-263 ranked among the most highly expressed miRNAs in both Ae. aegypti and An. stephensi . aga-bantam, aga-miR-263a, aga-miR-8, aga-miR-10, aga-miR-184 and aga-miR-281 were the most abundantly expressed miRNAs in An. gambiae . Thus, despite the differences in expression level, most of these reported abundant miRNAs in different mosquito lineages are still among the top 10 most highly expressed miRNA in An. sinensis.
Comparisons between egg and larva, larva and pupa, and pupa and unfed adult female are shown in Figs. 3 and 4. A majority of miRNAs were differentially expressed in the four stages. For example, 41 miRNAs were up-regulated and another 33 down-regulated between the embryo and larva stage. Especially, asi-miR-2943 and asi-miR-286b were up-regulated more than tenfold change. While between the larva and the pupa, 22 miRNAs were up-regulated and 20 other miRNAs were down-regulated. We also identified 15 significantly up-regulated miRNAs and 22 significantly down-regulated miRNAs from the pupal to the adult stage. And asi-miR-1891 was significantly down-regulated in this process. Dramatic alternation of miRNA expression during these three stages signifies that these miRNAs may be involved in the regulation of metamorphosis during development. What's more, asi-miR-2943 was exclusively expressed in the embryo stage, which is also highly expressed in the embryos in both Ae. aegypti, An. stephensi  and An. anthropophagus , indicating its important roles in embryogenesis across many mosquito species. However, miR-2943 was present at low levels in both Cx. quinquefasciatus and Ae. albopictus C7/10 cells . Similarly, miR-1891 was most abundantly expressed in Ae. aegypti and An. stephensi pupal stage , however, asi-miR-276-3p was the highest expressed miRNAs in An. sinensis pupae, although asi-miR-1891 also exhibited a high expression. These results indicate that, on the one hand, miRNA profiles of mosquito lineage may differ from each other, one the other hand, specifically expressed miRNAs or regulatory miRNAs during developmental stages may also be different. In addition, sample preparation, detection methods and sensitivity may also lead to inconsistency between different studies.
Moreover, we observed that the expression level of miRNAs did not correlate with the fold change. Some miRNAs showed higher expression but with low fold changes. For instance, asi-miR-276-3p, asi-miR-1, asi-miR-100 and asi-miR-7 were among the top 10 most highly expressed miRNAs in all developmental stages but showed low log2-fold changes. However, asi-miR-193 and asi-miR-nov54 were low expressed miRNAs in An. sinensis developmental stages but showed high log2-fold changes. Additionally, asi-miR-309a, asi-miR-2943, asi-miR-315-3p and asi-miR-iab-4-3p could not be detected in the pupae and larvae libraries, asi-miR-275-5p and asi-miR-2491-3p was not identified in the pupae and adult females' libraries, and asi-miR-980-3p was not found from the pupae library. These results indicate that the expressions of some miRNAs are probably stage specific. We assumed that stage-specific miRNAs might be involved in regulation of embryogenesis, organ differentiation, growth, and reproduction during a specific developmental stage. Investigation on stage-specific miRNAs may detail understanding of mosquito biology and could provide mosquito-specific targets for disease control. However, the function of majority of the miRNAs remains to be determined.
To confirm our sequencing results, we performed quantitative real-time PCR analysis using the RNA samples used for small RNA NGS. Thus, nine miRNAs that showed dramatic changes in expression level at different developmental stages were selected for validation. All nine miRNAs (asi-miR-276-3p, asi-miR-2943, asi-miR-1891, asi-miR-286b, asi-miR-1175-3p, asi-miR-263a-5p, asi-miR-nov23, asi-miR-nov54, and asi-miR-nov55) could be detected in the sample. Six miRNAs showed similar expression patterns as those revealed by our sequencing analysis, which indicated a low false discovery rate of our sequencing data and supported the validity of the profiling. However, the expression levels of asi-miR-263a-5p, asi-miR-nov23, and asi-miR-nov55 detected by qRT-PCR were inconsistent with that of our sequencing results. We inferred that this might be caused by the experimental conditions, such as base bias, amplification efficiency, and analytical error in both small RNA NGS and the qRT-PCR, but some other unknown reasons cannot be excluded.
Mosquitoes grow through four phases of metamorphosis: egg, larva, pupa and adult. Knowing the different stages of the mosquito's life will help us to prevent mosquitoes and disease transmission. To further understand the role of miRNAs in An. sinensis, miRNA target gene prediction was performed based on miRNA/mRNA interactions to provide molecular insight into their function by using two computational methods. A total of 7198 annotated mRNA transcripts were predicted as putative target genes for 95 differentially expressed miRNAs. The enriched biological functions and signaling pathways with significant role at a specific stage of development were predicted by GO term annotation and KEGG Pathway analysis. Metamorphosis from embryo to larval and from pupal to adult stage involves biological functions such as cellular component, membrane, cytoplasm and organic substance transport. In addition, enriched pathways involved in Protein processing in endoplasmic reticulum, Propanoate metabolism, Folate biosynthesis, Citrate cycle (TCA cycle) and Valine, leucine and isoleucine degradation were found. These biological functions and pathways might be important for degeneration and formation of tissue/organ, which happens during immature stages to complete its transition from egg to adult.
Network of miRNA-mRNA interactions was predicted by using results from miRNA analysis and mRNA retrieved from developmental genes of An. gambiae and then visualized by Cytoscape. Further analyses indicated that a number of miRNAs (asi-miR-317, asi-miR-2944a-5p, asi-miR-263b-5p, asi-miR-263a-5p, and asi-miR-2943) target a cluster of mRNA and vice versa. The candidate targets mentioned here have been reported to be key components in developmental pathways . For example, AGAP002352, one of the targets of asi-miR-263b-5p, is an annotated apoptosis-related gene (p53) in Ae. aegypti and many other mosquitoes. p53 plays an important role in programmed cell death in relation to development . Also, AGAP003108 (htl), AGAP001867 (hep) and AGAP005245 (lola) were the common target of asi-miR-281-5p. htl is one of two FGF (Fibroblast Growth Factor) receptors, and FGF signaling regulates a variety of biological processes during development, such as cell differentiation and cell proliferation . hep is one component of Ras signaling pathways which also could regulate many developmental processes . lola, which is called axon guidance gene that regulate wiring of the olfactory system in D. melanogaster , have been reported to have many orthologs in mosquito . In addition, knock-in and knock-down of specifically and temporally expressed miRNAs in Ae. albopictus by microinjection have improved our understanding of regulated miRNAs in mosquito development . Intervention on aal-miR-286b led to a profound decrease in the hatching rate of embryos. In addition, eclosion rate of larvae in aal-miR-2942 knocked-down group showed a significant decrease compared to control group. A marked reduction in longevity and fecundity was also observed in the miR-1891 inhibitor group. These research findings have established the exact functional roles these developmental genes and miRNAs in regulating the stage-specific development. However, additional extensive experimental analyses are required to confirm the interactions of miRNA-gene regulatory role in many phenotypic changes during developmental stages in further ongoing research work.
Although miRNAs are reported to play a role in development in C. elegans, fruit flies and other insects, and significant stage-specific expression was observed for a number of miRNAs in various mosquito species. However, the role of miRNAs in metamorphosis and development is far less understood and remains an enigma. For some specific or novel miRNAs, their functional roles still need to be confirmed by biological experiments. Furthermore, only very limited specific target genes have been identified for small part of miRNAs. It is important to note that the result in this work is a key step towards improving our understanding of the complexity and regulation mode of miRNAs in An. sinensis embryogenesis and metamorphosis. Further analysis of stage-specific miRNA expression and functions would be helpful to decipher the complex genetic network that controls mosquito at a crucial stage.
Anopheles sinensis is the most common and wide-distributed malaria vector exhibiting a variety of ecological behaviors and feeding habits throughout the China. It is also of great medical and veterinary importance. Thus, identification of key miRNAs required for gene regulation throughout the life cycle will not only help to a better understanding of its developmental biology but also may help to conceive novel approaches to control this vector. However, the function and molecular mechanism of these miRNAs are still need to be further investigated.
In conclusion, this study provides a comprehensive account of miRNA profile and its dysregulated expression patterns across four different stages of An. sinensis development using the high throughput sequencing. Several miRNAs show stage-specific expression pattern in particular developmental stages. We also investigate targets of these dysregulated miRNAs and their related functions in important life activities in order to provide a better understanding of mosquito development. In turn, such information would provide the basis to conceive novel approaches to control this vector in future.
XYF conceived and designed the study, XYF and WH performed the experiments. XYF and HW analyzed the data and drafted the manuscript. JWW and SSZ critically revised the manuscript. All authors read and approved the final manuscript.
The authors would like to thank Dongxiao Yang from the School of Bioinformatics, Nanjing Agricultural University for providing the computational algorithm involved in the work. This work was supported by the National Natural Science Foundation of China (Nos. 81271867 and 91431104), International Science and Technology Cooperation Program of China (No. 2014DFA31130) and National Science and Technology Major Program (No. 2009ZX10004-302). This work was also supported by Shanghai Municipal Commission of Health and Family Planning Grant (No. 20174Y0089).
The datasets supporting the conclusions of this article are available in the NCBI BioProject repository, [PRJNA412443, this Sequence Read Archive will be released on 2018-10-01 or upon publication].
13578_2018_227_MOESM1_ESM.xlsx Additional file 1: Table S1. Primers used in qRT-PCR validation.
13578_2018_227_MOESM2_ESM.xlsx Additional file 2: Table S2. Novel miRNAs identified in four life stages libraries of An. sinensis.
13578_2018_227_MOESM3_ESM.xlsx Additional file 3: Table S3. Target gene candidates for known and novel miRNAs in An. sinensis.
13578_2018_227_MOESM4_ESM.xlsx Additional file 4: Table S4. Developmental target gene candidates for 32 key miRNAs during development in An. sinensis.


55 Glass Los Angeles is centrally located in the wholesale district, 3 miles south of downtown. Our only business is glass supply, not installation or contracting, and for that reason we are able to offer exceptionally low, wholesale pricing.
55 Glass is able to custom fabricate virtually any type of flat glass for indoor and outdoor applications. In addition, 55 Glass has 55 stock items of glass table tops. We offer short lead times and same day service on some glass products.


text:
package com.fundynamic.d2tm;

import com.fundynamic.d2tm.game.entities.entitiesdata.EntitiesData;
import com.fundynamic.d2tm.game.scenario.factory.AbstractScenarioFactory;
import com.fundynamic.d2tm.game.scenario.factory.IniScenarioFactory;
import com.fundynamic.d2tm.game.scenario.factory.RandomMapScenarioFactory;
import com.fundynamic.d2tm.graphics.ImageRepository;
import org.junit.Assert;
import org.junit.Test;

import static org.mockito.Mockito.mock;

public class GameTest {

    @Test
    public void returnsRandomMapScenarioFactoryWithNoFileName() {
        Game myGame = new Game("my Game", "");

        AbstractScenarioFactory scenarioFactory = myGame.createScenarioFactory(mock(ImageRepository.class), new EntitiesData());

        Assert.assertTrue(scenarioFactory instanceof RandomMapScenarioFactory);
    }

    @Test
    public void returnsExpectedScenarioFactory() {
        Game myGame = new Game("my Game", "my-awesome-map.ini");

        AbstractScenarioFactory scenarioFactory = myGame.createScenarioFactory(mock(ImageRepository.class), new EntitiesData());

        Assert.assertTrue(scenarioFactory instanceof IniScenarioFactory);
    }
}

redpajama_set_name: RedPajamaGithub


text:
Saku City, Nagano Prefecture The online migration salon "Remote City Hall" is celebrating its 2nd anniversary today! Released a special radio drama at FM Remote City Hall
Saku City, Nagano Prefecture
The online migration salon "Remote City Hall" is celebrating its 2nd anniversary today! Released a special radio drama at FM Remote City Hall
Mikiha Spira Spica and Takumi Natsume will join the main cast, and attractive spots in Saku City such as the city's ski resort Parada and the supermarket Tsuruya will also appear.
From January 2021, Saku City, Nagano Prefecture (Mayor: Seiji Yanagida) will open an online migration salon "Remote City Hall" using Slack operated by the local government for the purpose of increasing migration, settlement and related population, and improving civic pride. " was launched. In commemoration of the second year today, we will release a special project radio drama "Hoshi no Saku Yoru ni" at FM Remote City Hall.
[Image 1
FM Remote City Hall Radio drama "Hoshi no Saku Yoru ni" can be viewed from the following URL.
https://anchor.fm/fm-remote-cityoffice/episodes/ep-e1tu5kg
■ Purpose of this project
"Remote City Hall" in Saku City, Nagano Prefecture is a digital community that was born against the background of the corona disaster, and is the first local government Slack-based migration online salon. It has attracted attention as a measure aimed at increasing the digital "related population" as well as the immigrant and permanent population, and has now grown into a platform with more than 2,100 participants.
Today, in commemoration of the second year since the start of the remote city hall, we released the radio drama "Hoshi no Saku Yoru ni ni" set in Saku City, which was completely produced by Mr. Yano Tetsu. This work is set in various real-life spots such as the ski resort "Parada" in Saku City and the supermarket Tsuruya. While synchronizing with the main character's feelings, we aim to let you know the charm of Saku City and feel closer to it.
Mr. Tetsu Yano, Manager of Remote City Hall
One of the attractions of my favorite city, Saku City, was "Isn't it possible to appeal to the beautiful starry sky!?"
The original work itself was written by me about 20 years ago, but since the "star" was an important element, I decided to turn it into a radio drama set in Saku City. .
I completed the story while remembering the perfect starry sky from the Arafune Panorama Camp Field that I actually saw last September, so I think it's a drama where you can feel the starry sky of Saku City from each description. . It is also recommended for those who have never listened to radio dramas, so please darken your room and envision the wonderful starry sky of Saku City while listening to it! Katsue Handa Assistant Manager Remote City Hall
I have been watching the starry sky in Saku City since I was a child. I feel that the perfect starry sky from the magic hour at dusk, the Milky Way that can be seen clearly at any time, and the splendor of a natural planetarium are truly treasures of Saku City.
Many people probably have a favorite place to go when they want to see the stars.
This time, a wonderful story about the wonderful starry sky of Saku City was born, and I hope that many people will listen to it. Don't forget to listen to the names of shops and spots that come up from time to time!
Special radio drama "Hoshi no Saku Yoru ni" as Mami / Spira Spica Mikiha [Image 5
Whoo! Spira spica Stem Leaf! This is the first time I have tried a radio drama. Mami and I have some similarities, and it was a lot of fun acting out each line.
In addition, various spots in Saku City appear in the story. I wanted to go to Saku City to see what it looked like and what it tasted like! Spira Spica has a lot of star songs, so I'd like to do a live performance under the starry sky in Saku City someday. It's a wonderful story that reminds us of the pure feelings we had forgotten about, "On a starry night." I hope you all enjoy the miracle that happened one night.
Spira Spica (abbreviation: Spipi) is a solo project by Mikiha, an innocent smiley heroine who naturally makes the viewers and listeners smile. Debuted in 2018 with "Start Dash" with pure pop rock. In 2022, the opening theme "Sansan Days" for the TV anime "Sono Kisekae Ningyo wa Koi wo Suru" will become a hit song. With their characteristic sunshine-like brightness, their music that encourages listeners' hearts and their live performances that are full of fun and happiness have become a hot topic.
Special radio drama "Hoshi no Saku Yoru ni" Shaw / Takumi Natsume [Image 6
[Automatically generated description of a boy in a white shirt] It was my first time doing voice work, and I had a lot of fun recording it. At first, I was planning to record by myself, but if it's nice to meet you, Mikiha-san, I'd like to record a conversation! It is partially recorded simultaneously.
I think Saku City in Nagano Prefecture is a wonderful city with an abundance of nature and clean air. I hope to hear from you soon. A small miracle that happened under the starry sky.
Please take a look at the starry sky in Saku City and listen to it! 【profile】
She made her stage debut at the age of 8, and as a child she played Seryozha in Anna Karenina and Young Simba in The Lion King. After taking a hiatus to concentrate on his studies, his major stage works in recent years include "PERSONA5 the Stage", "GOYA" and "Cinderella Story". In the future, we will expand the range of activities regardless of genre.
■ Synopsis of the radio drama "Hoshi no Saku Yoru ni"
The main character Mami's favorite place is a parking lot on a hill where the night view and stars can be seen beautifully.
At first, he is wary of the suspicious behavior of a young man, Sho, whom he sees for the first time. And the reason is to fulfill a promise with a childhood friend who was separated when he was a child. The situation develops rapidly due to the thoughts of a man with such a pure heart.
A town full of stars ☆ What is a small miracle that happened in Saku? Radio drama "Hoshi no Saku Yoru ni" viewing URL:
FM Remote City Office: https://anchor.fm/fm-remote-cityoffice ■Overview
・Title: In the Starry Night
・Director/Screenplay/Director: Yano Tetsu
・ Cooperation: Katsue Handa
・Performers: Spira Spica Mikiha (role of Mami), Takumi Natsume (role of Shaw), Saku (role of Misaki)
KANA (grandmother, narration)
・Facilities in Saku City where the stage was set
1. Nagano Ranch 1549-1 Shinkoda, Saku City, Nagano Prefecture 385-0007 2. Sakudaira Highway Oasis "Palada" 2681 Shimohirao, Saku City, Nagano Prefecture 385-0003
3. JAXA Usuda Space Center 1831-6 Kamiotagiri, Saku City, Nagano Prefecture 384-0306
4. JAXA Mizasa Deep Space Exploration Ground Station 1905-43 Tateshina, Maeyama, Saku City, Nagano Prefecture 385-0046
5. Usuda Star Dome 3113-1 Usuda, Saku City, Nagano Prefecture 384-0307 6. Tsuruya 3020-1 Otsuka, Nakagomi, Saku City, 385-0051
7. Bakery Rian 137-1 Hara, Saku City, Nagano Prefecture 385-0052 -reference-
■ What is Saku City, Nagano Prefecture?
Saku City is surrounded by beautiful mountains such as Mt. Asama, Mt. It takes about 75 minutes to Tokyo on the JR Hokuriku Shinkansen and about 110 minutes from the Tokyo metropolitan area on the Joshinetsu Expressway, making it highly convenient for high-speed transportation. The area boasts one of the highest percentages of fine weather in Japan, with little rainfall throughout the year and little snow in winter. In addition, no tropical night has ever been observed in the history of meteorological observation, making the climate extremely comfortable to live in. We have advanced community medicine and a comprehensive care system nationwide, and medical welfare is substantial.
What is Remote City Hall?
"Remote City Hall" is a new migration platform that uses the business chat "Slack" to respond to migration and living consultations. We are promoting real-world information on Saku City and immigration, as well as casual information exchange with citizens. In addition, we hold idea discussions on the issues of Saku City and immigration, and work to realize ideas that lead to problem solving. In July 2021, we announced "Shijuly," a trial housing support and subsidy service conceived and planned from posts in the remote city hall. In August 2021, we will solicit applications from remote city hall participants and certify remote city hall section chiefs and employees for fiscal 2021, and will activate communication centered on content such as the new project "FM Remote City Hall" centered on section chiefs. I have been there. Through these activities, the remote city hall won Gold in two categories of PR Awards Asia, Golden Worlds Awards, City Promotion Awards Gold Award / Future Creation Award, PR Award Grand Prix Bronze, Japan Marketing Award Encouragement Award, Awarded ACC Silver in the ACC TOKYO CREATIVITY AWARDS Branded Communication category (C category: PR).
HP: https://www.city.saku.nagano.jp/outside/citypromotion/salon Slack: https://saku-remote-cityhall.slack.com
Twitter: https://twitter.com/SAKUonlinesalon
■What is Slack?
Slack is a pioneering channel-based messaging platform. We have revolutionized the way business communication is done. Hundreds of companies are now using Slack to align their teams, get the systems they use in one place, and move their business forward. Only Slack provides a secure, enterprise-grade environment for the world's largest companies.
Slack is a whole new layer in the world of business technology. A place where we can "work together" more efficiently, have all the software tools and services we use in one place, and easily find the information we need. Slack is the center of your work.
HP: https://slack.com/
Official image material: https://brandfolder.com/slack
freee It is now possible to send bulk e-mail questionnaires regarding system responses to invoice business partner management business partners
Bushiroad Music Co., Ltd. From the D4DJ project, Call of Artemis 1st Album "Call of Artemis" and "D4DJ Groovy Mix Cover Tracks vol.7" are released today!
4 Nov 22 rehow
11/5 The "Temmabashi Hachikenya Waterside Hagukumi Project", which aims to create a community where parents and children can nurture each other, will hold a "Hagukumi Marche"!
General Incorporated Association U-me [11/5] The "Temmabashi Hachikenya Waterside Hagukumi Project", which aims to create…
Sony Music Entertainment Sony Music's VTuber project "VEE" and the first "Apex Legends" custom tournament "Koha CUP" hosted by VTuber Kohaku Akiyuki will finally start at 19:00 on November 26th!
Sony Music Entertainment Inc. Sony Music's VTuber project "VEE" and the first "Apex Legends" custom…
13 Oct 22 rehow
Nitta Gelatin Co., Ltd. Exhibited at "FOOD STYLE Japan 2022", one of Japan's largest food business exhibitions covering the food service, ready-made meal, and retail industries.
Nitta Gelatin Co., Ltd. Exhibited at "FOOD STYLE Japan 2022", one of Japan's largest food…

redpajama_set_name: RedPajamaCommonCrawl


best lighting for vegetative growth whether youre propagating tabletop lettuce or a batch of the sticky ickies window sills just wont cut it you need yourself a proper lighting rig to get best lighting for vegetative growth of marijuana.
best lighting for vegetative growth marijuana best lighting for vegetative growth of marijuana.
best lighting for vegetative growth for those who love the p600 but dont have quite that large of a budget or size of a grow tent the advanced platinum series p300 is a great runner up best lighting for vegetative growth of marijuana.
best lighting for vegetative growth cannabis in its flowering stage how to grow weed with cfls grow weed easy.
best lighting for vegetative growth marijuana plants in the vegetative stage best lighting for vegetative growth of marijuana.
best lighting for vegetative growth using t5 grow lights for cannabis best lighting for vegetative growth.
best lighting for vegetative growth acke led panel grow light plant light pcba hydroponic grow lightled grow best lighting for vegetative growth of marijuana.
best lighting for vegetative growth led light emitting diodes best lighting for vegetative growth.
best lighting for vegetative growth plants in pots five things to think about when choosing a container best lighting for vegetative growth.
best lighting for vegetative growth manipulating the color spectrum best lighting for vegetative growth.
best lighting for vegetative growth professional led grow lights top best hydroponic grow lights compare buy save heavycom best hydroponic grow lights cannabis grow lights marijuana growing.
best lighting for vegetative growth best lighting for vegetative growth of marijuana.
best lighting for vegetative growth companies that manufacture led grow lights have a lot of different ideas about what types of light are needed during the growth process and while weve best lighting for vegetative growth of marijuana.
best lighting for vegetative growth a 62 light schedule could increase plant growth the best marijuana grow lights cannabis light cycle.
best lighting for vegetative growth led growing lights cannabis best lighting for vegetative growth.
best lighting for vegetative growth bud grown with 400w hps best lighting for vegetative growth.
best lighting for vegetative growth t5 grow lights what you need to know best lighting for vegetative growth.
best lighting for vegetative growth best lighting for vegetative growth.
best lighting for vegetative growth led grow lights and marijuana how does color spectrum affect growing weed grow weed easy.
best lighting for vegetative growth vegetative stage best lighting for vegetative growth.
best lighting for vegetative growth best t5 grow lights best lighting for vegetative growth.
best lighting for vegetative growth clonesx20fromx20bolderx20cannabisx20 best lighting for vegetative growth of marijuana.
best lighting for vegetative growth robofarm 15w t5 led seedling vegetative 4 grow light the best marijuana grow lights best grow lights for indoor marijuana growing.
best lighting for vegetative growth marijuana weed cannabis best lighting for vegetative growth.
best lighting for vegetative growth led vegetative grow light grow light from best lighting for vegetative growth.


Unique custom home in popular neighborhood. Two bedrooms have baths. Large master suite is the upper level with a private balcony and a new jetted tub plus an office area. Beautiful vigas in the great room and lots of Saltillo tile. Corner kiva fireplace. Discover nooks and crannies, angles and shelves for all of your storage and display needs. Enjoy outdoor living on the covered patio or gazebo.
Incredible home and uniquely Southwest with seldom seen features. Private master with awesome master bath and private deck. Kitchen for the pros.
San Juan Country Club unique custom features, clerestory windows, 14'7' entry ceiling, master bedroom suite with sitting area to a covered patio. Must See!
Beautiful home in the Country Club. Large Kitchen,Open floor plan Landscaped front and back yards. Fireplace,Office area,Formal dinning area.Nice fences and more! Don't miss out, show your buyers today! See private remarks.


Your skin changes with the seasons; these are the naturally based products you need to look refreshed and ready for spring.
Get to know Posh Consultant Laura Osminski and her favorite pampering products.
Meet Pink + 2 Jessica Christine Vezino, one of our amazing Consultants who joined in October 2017.


They're white and puffy and full of personality! What are we talking about?
We're talking about our beautiful Bichon Frise puppies for sale. You've definitely seen these little beauties because they command attention from the moment you set your eyes on them. The American Kennel Club has a standard description calling them "a white powder puff of a dog whose merry temperament is evidenced by his plumed tail carried jauntily over the back and his dark-eyed inquisitive expression." That sounds about right to us! Continue reading to learn even more about Bichon Frise puppies for sale.
Okay, so there's one thing we can say for sure about the Bichon Frise and it's that they are easy to train when it comes to tricks and commands. Isn't that exactly what you want to hear? They love to please and respond well to positive reinforcement and treats. Bichons like to bark, we have to be honest, so when training them keep this in mind. This behavior can be unlearned. Conversely, Bichons are difficult to house train. Crate training is recommended for them.
We talked about their bright white coats, but let's go a bit further. Did you know that Bichons are hypoallergenic? This means people who suffer from dog allergies can take home any of our Bichon Frise puppies for sale. They shed very little and they have coats that are very similar to Poodles. As their hair grows, it gets curlier and curlier making them even more adorable, if that's possible. To keep them looking beautiful, professional and regular grooming is necessary to maintain their powdery white coats.
They are known to be hyper and excitable but Bichon Frise puppies don't really require a lot of exercise. Of course, for any dog physical activity is good for them, but they don't need as much as say Boxers. One or two daily walks a day is perfect for them, along with some play time around the house. Speaking of around the house, if you have kids at home, you'll find that Bichons get along well with them and other pets, as well. They can live in an apartment or large home, as long as they're loved and treated well, they'll be fine in any living environment.
If you're interested in purchasing one of our Bichon Frise puppies for sale, contact us today for more information. We know you'll enjoy these bundles of joy!


text:
Charlotte County Committee Vacancies
Home News detail
CHARLOTTE COUNTY, Fla. (Jan. 19, 2022) – The Affordable Housing Advisory Committee is searching for one volunteer to represent banking/mortgage banking and to fill the unexpired term of Jim Kelley. The term begins immediately and expires Nov. 10, 2023.
The following MSBU/TU Advisory Boards are seeking volunteers who must be both a property owner within and reside within the Unit. Submit applications to Public Works Department, 7000 Florida Street, Punta Gorda, FL 33950; call 941-575-3600 or e-mail MSBU-TU@CharlotteCountyFL.gov.
Boca Grande Street & Drainage Unit is seeking one member to fill a vacant unexpired term through Oct. 31, 2024.
Buena Vista Waterway Unit is seeking two members to fill vacant unexpired terms through Oct. 31, 2024.
Edgewater North Waterway Unit is seeking one member to fill a vacant unexpired term through Oct. 31, 2024.
Greater Port Charlotte Street & Drainage Unit is seeking one member to fill a vacant unexpired term through Oct. 31, 2023.
Grove City Street & Drainage Unit is seeking one member to fill a vacant unexpired term through Oct. 31, 2022.
Gulf Cove Street & Drainage Unit is seeking one member to fill a vacant unexpired term through Oct. 31, 2024.
Gulf Cove Waterway Unit is seeking one member to fill a vacant unexpired term through Oct. 31, 2022 and Oct. 31, 2024.
Harbour Heights Street & Drainage Unit is seeking one member to fill a vacant unexpired term through Oct. 31, 2024.
Harbour Heights Waterway is seeking one member to fill a vacant unexpired term through Oct. 31, 2022 and Oct. 31, 2024.
Manchester Waterway Unit is seeking one member to fill a vacant unexpired term through Oct. 31, 2024.
Mid-Charlotte Stormwater Unit is seeking two members to fill vacant unexpired terms through Oct. 31, 2024.
Northwest Port Charlotte Waterway Unit is seeking one member to fill a vacant unexpired term through Oct. 31, 2023.
Pirate Harbor Waterway Unit is seeking one member to fill a vacant unexpired term through Oct. 31, 2024.
Rotonda Heights Street & Drainage Unit is seeking two members to fill vacant unexpired terms through Oct. 31, 2024.
Rotonda Lakes Street & Drainage Unit is seeking two members to fill vacant unexpired terms through Oct. 31, 2022.
Rotonda Sands Street & Drainage Unit is seeking one member to fill a vacant unexpired term through Oct. 31, 2022
South Burnt Store Street & Drainage Unit is seeking one member to fill a vacant unexpired term through Oct. 31, 2022.
South Charlotte Stormwater Unit is seeking one member to fill a vacant unexpired term through Oct. 31, 2022.
South Gulf Cove Street & Drainage Unit is seeking one member to fill a vacant unexpired term through Oct. 31, 2024.
Suncoast Waterway Unit is seeking five members to fill vacant unexpired terms through Oct. 31, 2022, Oct. 31, 2023, and Oct. 31, 2024.
**Note: THIS IS NOT A LEGAL ADVERTISEMENT AND SHOULD NOT BE USED AS SUCH.
If you require special assistance or accommodation, please include this information with your resume.
County Warns Public about 'Spoofing' Scam
Carmalita Park Softball Restrooms are Closed
Higgs Park Installation of Drinking Fountain
Charlotte County Government Calendar
**CORRECTION** Traffic Advisory - Lane Shift on Quesada Avenue
Charlotte Harbor Community Redevelopment Agency Meeting Jan. 25


text:
Construction›
Key facts about dams in the U.S. 2020
Published by Raynor de Best, Nov 16, 2020
Of the over 91,000 dams in the United States, only three percent were dams with hydroelectric facilities. Texas is home to the greatest number of dams in the U.S. As of 2019, there were over 7,000 dams in the Southern state.
Selected figures about dams in the United States as of 2020
November 2019 to January 2020
Dams and hydropower in the U.S.
Hydroelectric power generation in the U.S. 2019
U.S. hydropower capacity 2000-2018
Hydropower capacity in the U.S. 2018, by key state
Hydropower consumption in the U.S. 2006-2019
Raynor de Best
Research expert covering finance, insurance & real-estate, internet & e-commerce
Statistics on "Dams and hydropower in the U.S."
Global hydropower installed capacity 2014-2019
Hydropower global capacity in key countries 2019
Global hydropower production 2010-2018
Largest hydropower producing countries 2019
Global hydropower consumption 2000-2019
Largest hydropower consuming countries 2019
Countries with the highest share of hydropower consumption 2019
U.S. dam ownership share 2020
Number of dams in the U.S. 2019, by key state
Oldest hydroelectric facilities in the U.S. 2020
Hazard risk of dams in the U.S. 2020
Number of incidents at dams in the U.S. 2010-2020
Number of conventional hydropower plants in the U.S. 2016-2020
Largest hydropower plants by capacity in the U.S. 2020
Hydroelectric capacity additions in the U.S. 2009-2018
Conventional hydroelectricity production in the U.S. 2020, by key state
Hydropower consumption in the U.S. 2018, by key state
Global hydropower sector job share by country 2019
Hydroelectric power generation industry employment in the U.S. 2001-2019
Hydropower capacity additions outlook in the U.S. 2020-2050
Capital costs for a hydroelectric power plant in the U.S. 2017-2050
Electricity generated in the United Kingdom (UK) using hydro 2005 -2017
Water storage volume in dams Australia 2020 by state
The world's largest reservoirs according to volume
U.S. hydropower facilities by operating mode 2015
U.S. employment in hydropower operations by occupation 2013
Global GHG emissions from reservoirs by emission type 2016
United States' installed small hydropower facilities by size 2011
Number of pumped-storage hydroelectric power plants in Italy 2003-2018
Volume of aluminum reservoirs exported globally by the UK 2010-2017
Value of aluminum reservoirs (capacity >300l) exported globally by the UK 2010-2017
Value of aluminum reservoirs capacity >300l imported globally by the UK 2010-2017
Designed capacity of dams in the GCC 2007- 2016
Industry revenue of "manufacture of tanks, reservoirs and containers" in Denmark 2012-2024
Industry revenue of "manufacture of tanks, reservoirs and containers" in Italy 2012-2024
Industry revenue of "mattress manufacturing" in the U.S. 2012-2024
Healthcare consumer spending per capita forecast in Angola 2010-2025
Real transportation consumer spending per capita forecast in Australia & Oceania 2010-2025
Real recreation & culture consumer spending per capita forecast in Costa Rica 2010-2025
Real household upkeep consumer spending forecast in Peru 2010-2025
USACE. (March 1, 2020). Selected figures about dams in the United States as of 2020 [Graph]. In Statista. Retrieved January 20, 2021, from https://www.statista.com/statistics/1181694/us-dams-key-facts/
USACE. "Selected figures about dams in the United States as of 2020." Chart. March 1, 2020. Statista. Accessed January 20, 2021. https://www.statista.com/statistics/1181694/us-dams-key-facts/
USACE. (2020). Selected figures about dams in the United States as of 2020. Statista. Statista Inc.. Accessed: January 20, 2021. https://www.statista.com/statistics/1181694/us-dams-key-facts/
USACE. "Selected Figures about Dams in The United States as of 2020." Statista, Statista Inc., 1 Mar 2020, https://www.statista.com/statistics/1181694/us-dams-key-facts/
USACE, Selected figures about dams in the United States as of 2020 Statista, https://www.statista.com/statistics/1181694/us-dams-key-facts/ (last visited January 20, 2021)


Observing Pol Pot at close quarters during the one and only official visit he ever made abroad, to China in 1975, Philip Short was struck by the Cambodian leader's charm and charisma. Yet Pol Pot's utopian experiments in social engineering would result in the death of one in every five Cambodians—more than a million people.
Cambodia 1975–1982 presents a unique and carefully researched analysis of the Democratic Kampuchea regime of Pol Pot and the Khmer Rouge (1975–79) and the early years of the People's Republic of Kampuchea (1979–89). When it was first published in 1984, the book provided one of the few balanced and reasoned voices in a world shocked by media reports of incredible brutality.
Between 1975 and 1979 the seemingly peaceful nation of Cambodia succumbed to one of the most bloodthirsty revolutions in modern history. Nearly two million people were killed. As head of the Khmer Rouge's secret police, Comrade Duch was responsible for the murder of more than 20,000 of them. Twenty years later, not one member of the Khmer Rouge had been held accountable for what had happened, and Comrade Duch had disappeared.
When the Khmer Rouge came to power in Cambodia in 1975, they inherited a war-ravaged and internationally isolated country. Pol Pot's government espoused the rhetoric of self-reliance, but Democratic Kampuchea was utterly dependent on Chinese foreign aid and technical assistance to survive. Yet in a markedly asymmetrical relationship between a modernizing, nuclear power and a virtually premodern state, China was largely unable to use its power to influence Cambodian politics or policy.
This handbook of slogans, interspersed with historical commentary and contextual analysis, describes the Khmer Rouge regime and exposes the horrific foundation upon which it constructed its reign of terror. On April 17, 1975, the Khmer Rouge seized power in Phnom Penh. In the three years, eight months, and twenty days of their government, they made a tabula rasa of Cambodian society and culture, forcing the people to evacuate the cities and move to the countryside.


text:
Home / politics / What I need from Buhari to defeat Audu – Wada
What I need from Buhari to defeat Audu – Wada
Eric Daminabo 14:35:00 politics
Ahead of the November 21 governorship election in Kogi State, Governor Idris Wada has declared that all he needed from the President Muhammadu Buhari-led Federal Government is a level playing field before and during the election in the state.
He made this demand while interacting with State House correspondents shortly after meeting behind closed doors with Buhari at the Presidential Villa, Abuja.
Wada, who is the flag bearer of the Peoples Democratic Party, PDP, in the election, will be
squaring up with a former Governor of the state, Alhaji Abubakar Audu – the All Progressives Congress, APC, governorship candidate.
The Governor expressed optimism that if a level playing field was created for the election, the people of the state would give him overwhelming support at the poll based on his performance in office.
His words, "I will tell the people of the state to keep faith because we have a transparent President.
"Provided a level playing field is created and security is tightened, any miscreant dealt with in the course of the election, people given the opportunity to express their will, I believe that on the basis of performance I have laid down over the last three-and-a-half years, our people will provide an overwhelming support for my candidacy.
"All we need is a level playing field."
While noting that he was working hard ahead of the next month election, Wada maintained that he was collaborating with security agencies to ensure that there is a secure and safe environment for the people of the state in the course of the campaigns and during the election.
What I need from Buhari to defeat Audu – Wada Reviewed by Eric Daminabo on 14:35:00 Rating: 5
Eric Daminabo
politics|


Business listing information: phone and fax number, opening hours, how to reach and other information. You can read the good or bad reviews about the bowers company from Columbia ALABAMA.
bowers located at US Alabama in Columbia city. Contact phone number: 8034002105. The exact address: Alabama in Columbia, 310 Greystone Blvd.Postal zip code: 29203.


For online orders, go to www.chicken-republic.com and click on "Order Online" or go directly to order.chicken-republic.com. Click on "Our Menu" option, then Select your Food Options. Click on "Add to bag". Once completed, review your order and click on "Place Your Order" to finish the process.
For over the phone orders, call us at 0803 901 2601 to speak to our representatives and they will assist you in placing your order.
Your order will not be processed unless it has been verified via telephone by one of our representatives.
2. What should I do if I have not received an order confirmation email?
If you haven't received an order confirmation email, please check the spam message in your email or give us a call at 0803 901 2601 to get your order confirmation number.
1. How fast do you deliver?
We deliver in less than 30 minutes from when you place your order! To check the status of your order, please call us on 0803 901 2601 or visit the 'My Account' and click on 'Order History' to view the status of your order.
You pay the point of delivery via Mobile POS or Cash! Our drivers do not carry more than ₦3,000 in cash so please have exact change for your order.
3. Is there a delivery fee or minimum purchase required to make an order?
Delivery fee charge is ₦300, but during weekdays we have a special delivery fee of ₦150. The minimum order amount is ₦1,200.
4. Should I tip the driver?
Our drivers do everything to make your delivery delightful, so tips are appreciated but not required.
We are currently delivering in Lagos and Abuja (Wuse & Maitama) between 8.30 am and 6 pm every weekday, and Saturday between 12 pm and 7 pm.
We'll be expanding to more areas soon!
1. I forgot my password, what should I do?
If you've forgotten your password, click on Log In on the Home Page. Enter your email address and click on "Forgot Password". Then follow the instructions to recover your password.
2. Where can I view my previous orders?
For your previous order, log in to My Account, then click on My orders to view history of previous orders. To reorder, click on the "Reorder".
Visit the 'Account Information' page on 'My Account' area. You'll see a checkbox saying "Change password" - if you check it, you'll find a form to change your current password.
1. What do I do if I have a problem regarding my order, food or delivery?
If you have problem with your order, food or delivery, please call 0803 901 2601 and our representative would be happy to help.
2. What if I need to cancel or modify my order after I have placed it?
You can cancel or modify your order during the confirmation process. One of our representatives will call you to confirm your order within 10 minutes of placing an order online or via telephone. No cancelations or modifications are accepted after the final confirmation call.
3. What should I do if my food arrived and something is not right?
If your food arrives and something is not right with your order, please send an email to order@chicken-republic.com or call our representative at 0803 901 2601.
4. What is Chicken Republic's refund policy?
Once the order has been confirmed and has been paid for, there is NO refund.
Did you get a text or an e-mail from Chicken Republic Online with an offer?
If its an immediate Discount Coupon Code, please insert the Coupon Code in the Shopping cart to deduct the amount!
If its a Lucky Customers Deal, please insert the Coupon Code in the Shopping cart and wait for your text message or e-mail to see if you won or not!
Note: For all orders, the minimum order amount is ₦1,200!
We aim to delight you! If you ever encounter an issue, please email us at order@chicken-republic.com or call us on 0803 901 2601.


Atlas Roll-Off has an outstanding disposal facility for people from across New York to use. We are centrally located in Brooklyn and our onsite debris dump and disposal facility provides garbage disposal services for residents and contractors in all 5 boroughs of New York. It is a the perfect place to bring down whatever debris and garbage you have from a recent construction or home project.
Our safe and efficient services include any non-hazardous demolition debris, rubbish and construction debris. Our transfer station facility provides easy access to all vehicles including your van, pick-up truck, cargo box truck or dump truck. Our aim is to make your trip to our dump go as smoothly and quickly as possible.
Our environmentally sound disposal facility in Brooklyn has been helping to keep New York clean for over 30 years. Do your part in keeping the place you live clean by bringing down old items to our dump. Every small part of helping the environment goes a long way. We are open six days a week, Monday-Friday from 6:30am to 4:00pm, and Saturday from 6:30am to 1:00pm for your best convenience. We look forward to seeing you and helping you dispose of any junk or debris you may have with you.
Note: the above list is not an all inclusive list of acceptable material. We do not accept any hazardous material of any kind or ammunition. So be sure to check what you are bringing to ensure we accept. If you have any questions or are unsure of what you can bring to the disposal facility, please give us a call at 718-345-6451. We will answer any questions or concerns you may have about our dump and the services we provide.
Atlas Roll-Off is proud offer our elite dump and disposal services for customers across the NYC area, including Manhattan, Brooklyn and Queens. Our commitment to excellent customer support allows us to provide you with the highest quality services. We understand the importance of being able to safely and efficiently dispose of your waste from demolition and other projects.
Our disposal services are great for assisting in construction and demolition jobs. Our professionally trained staff brings their years of knowledge and experience into every disposal job. We look forward to assisting you with your next project. For more information about our Brooklyn garbage dump and disposal facility, please contact us today. The number to call for our office is 718-345-6451. We will answer any questions you may have about any of the services we provide.


The facility, an ICM design, was erected by Fagen Construction in 2005 and went online in 2006. Its original nameplate specified plant capacity at 40 million gallons of ethanol per year. Today, Carbon Green BioEnergy produces at 55 million gallons yearly.
In June 2009, new investors purchased the plant and took steps to expand its potential. Everything was evaluated—processes, ingredients, equipment, and grain storage structure—in a holistic approach that considered where improved technology could offer better efficiencies and the best product. Then, in order to increase production and equip our facility for the convenience of corn producers who deliver to us, we made strategic improvements.
The plant, designed for a 2.75 (non-denatured) gallon yield of ethanol per bushel currently performs at an improved 2.85 (non-denatured) gallons per bushel. With every incremental upgrade, we also lessen the environmental impact of our production. Since 2009, we've reduced our water consumption by 25% and our energy consumption by 18%.


FIRST KARATE LESSON IS A FREE TRIAL!!
The next 2 classes can be paid individually at £5 each.
At United Sports Karate Organisation we offer you three great life membership options to our schools of excellence.
Both of these two options offer you or your child an affordable start in our superb system of Sport Karate. We aim to make you feel fitter and better about yourself and at the same time learn what could be a valuable form of self-defence.
A pair of karate sparring gloves.
We normally suggest trying it for 2 or 3 classes after the trial lesson before deciding whether to sign up for the membership or not.


Lakeshore cabin on over 2 acres on beautiful Shuswap Lake. This 2 bedroom cabin is in great shape and has recent renovations. The lot has over 100 feet of waterfront. No hydro bills here as its has its own creek that supplies power to the cabin through a pelton wheel. This off the grid community has much to offer including the white sand at Silver Beach Provincial Park, boating, fishing, snowmobiling, hunting and simply "getting away from it all". A very well kept secret location! All meas. approx.


text:
Home News Hungary's Orban meets Putin for nuclear talks
Hungary's Orban meets Putin for nuclear talks
Hungarian Prime Minister Viktor Orban meets Russian President Vladimir Putin on Tuesday for talks touching on Russia's expansion of the EU nation's only nuclear power plant.
The Kremlin said in a statement that the two close trade and energy partners were holding "substantive discussions" on building up the capacity of Hungary's Paks facility.
The statement said Paks was responsible for producing 40 percent of the energy consumed in the former Warsaw Pact state.
It added that Russia supplied about 80 percent of Hungary's oil and 75 percent of its natural gas.
"The negotiations with Hungary have reached their active stage," Russia's Rosatom state atomic energy corporation chief Sergei Kiriyenko told the Interfax news agency.
Hungary's Nepszabadsag daily reported that Budapest and Moscow were likely to sign an inter-governmental agreement on the plant's expansion during Orban's visit.
Paks is run by Hungary's state-run MVW — a group that also imports natural gas from Gazprom and would like to negotiate a price cut with the Russian energy giant.
The Hungarian parliament in 2009 approved a decision to add two more reactors to the four already operating at Paks.
France's Areva and US electic company Westinhouse along with Japanese and South Korean power suppliers had previously expressed interest in bidding for a contract of the Hungarian plant's expansion.
But the Nepszabadsag daily report said Russia's Rosatom was the only potential bidder willing to offer pre-financing.
Any deal with Russia could lead to domestic criticism of Orban because no formal bidding process for the plant's expansion has yet been launched.


How does one get the system to actually use the IPv6 Temporary Addresses?
Is there some trick to getting the system to actually use the IPv6 Temporary Addresses? On multiple system, some in a domain and some stand-alone, both before and after the Anniversary Update, the fixed IPv6 address is used after a few hours. Mucking with the network (e.g., turning temporary addresses off and on) gets the system to use a temporary address for a while, but check back in a few hours or the next day, and the permanent address the one being used. "ipconfig" shows multiple temporary IPv6 addresses available, but checking https://www.google.com/#q=what+is+my+ip in Edge, Chrome, and Firefox show the permanent address being used. "ping" can be convinced to use another address with the "-S", but otherwise it also selects the permanent address.
Am I the only person seeing this? Could it be some oddity of this network (Cox DHCPv6-PD through an EdgeRouter Lite)?
Having, but not using, temporary IPv6 addresses does not provide much in the way of privacy.
I will feedback this issue in our platform. Thanks for your comments on this issue.
In addition, as I known, Temporary addresses may be used as source address for originating connections, while external hosts use a public address by querying the Domain Name System.
Network interfaces configured for IPv6 use temporary addresses by default in OS X Lion or later Apple systems, and in Windows Vista, Windows 2008 Server or later Microsoft systems.
More specifically, each process generates a resource request and the OS delivers. The IPv6 SAA is defined in RFC 2462, but the "Temporary IPv6 Address" is because of Windows implementation of RFC 4941. So the question becomes what process is responsible for initiating an OS request to open a socket that has these Privacy Extensions.
There are temporary addresses, but since none of the web browsers or even ping like to use them after a few hours (perhaps until a second temporary address is generated?), they don't seem to be terribly useful.
If Edge should not be using a temporary IPv6 address, then why does it sometimes do so (e.g., for a while after the network stack has been reset)?
I am seeing the same behavior on Windows 10 Home.
Initially browsers use the temporary IPV6 address. After a while they start consistently using the permanent one. I do think this is a bug but it appears few others have even noticed this behavior.
Ping does the same thing.
On the plus side the 64 bit ipv6 interface identifier that windows 10 generates is not based on the Ethernet MAC address.
This is still a problem in 16299.64. What is the point of having temporary IPv6 addresses if they are often not used? After a reboot, the temporary address works as it should, but when a new temporary address is added, everything goes back to using the permanent address.
All the "do not track", Ghostery, Privacy Badger, tools in the world are of little help if the lower 64 bits of the computer's address is always the same and pretty much guaranteed to be unique.
Making privacy promises and then not keeping them is arguably not a security vulnerability, but it is very close to one.
Still a problem in 2018, a year and a half later. I am on Windows 10 version 1709, build 16299.192. ipconfig shows multiple addresses but a "what's my ip search" shows the permanent address.
My only solution so far - rebooting. Yes after a reboot Windows starts using the temporary address. Not exactly convenient though.
This seems to have been fixed sometime between build 16299 and 17127. I'll keep checking, but so far I haven't seen the problem since I switched to the slow ring insider builds.


ARLINGTON, VA.--Womble Bond Dickinson telecom lawyer Erin Fitzgerald will discuss broadband issues in rural America at the upcoming 2018 Washington Conference of the National Association of Development Organizations (NADO) and the Development District Association of Appalachia (DDAA).
Fitzgerald will be joined in the panel discussion, titled "The Broadband Gap: Response and Opportunity," by NTCA's Josh Seidemann and Doug Brake, Director of Telecommunications Policy with the Information Technology and Innovation Foundation. The panel will discuss recent developments at the federal and state levels to promote broadband, the Trump Administration's infrastructure proposal and the role of local stakeholders in broadband development.
The National Association of Development Organizations (NADO) provides advocacy, education, research, and training for the nation's regional development organizations. The association and its members promote regional strategies, partnerships, and solutions to strengthen the economic competitiveness and quality of life across America's local communities.
The Development District Association of Appalachia (DDAA) is a membership organization of the 73 Local Development Districts (LDDs) serving the 420 counties of the Appalachia Region.
The 2018 NADO-DDAA Washington Conference takes place March 19.
Erin Fitzgerald advises telecommunications carriers, broadband service providers, and technology companies on complex commercial and regulatory matters. Erin also serves as Regulatory Counsel to the Rural Wireless Association (RWA), and has extensive experience on issues regarding privacy/data protection, spectrum auctions, universal service, broadband deployment, data roaming, network neutrality, and wireless licensing. Erin advocates in rulemaking and policymaking proceedings on behalf of RWA as well as individual clients, and frequently appears before the Federal Communications Commission (FCC).


Hurray! I'm not the only one! Unfortunatly it appears that I am at the begining of a very long (and expensive) road.
Andrew from Vuurwapen Blog has had a similar experience with his own Kimber 9mm 1911. Since purchasing his he has replaced so many parts that the pistol appears to be a Kimber in name only.
Yup. I bought one once. NEVER again. Kimber either has to tight tolerances or a design flaw.
At their prices it's shameful.


text:
Federal Circuit Overrules COFC's "Narrowly Targeted" Review Standard; Corrective Action Challenges Just Became a Lot Harder
By: Hopewell Darneille
As we have discussed, a significant percentage of bid protests end up in an agency taking some form of corrective action, either entirely voluntarily based upon the agency's internal assessment, or as the result of external pressures – formal or informal – from the Government Accountability Office (GAO), the Department of Justice or a Court. A critical issue in such situations is the scope of the corrective action, and the extent to which the parties to the procurement, and particularly the original awardee, can challenge the same if the proposed corrective action appears to be going too far.
In an important new decision, the United States Court of Appeals for the Federal Circuit (the "Federal Circuit") has overturned a line of decisions in which the U.S. Court of Federal Claims (COFC) had ruled that an agency's proposed corrective action must be "narrowly targeted" or "tailored" to that which is necessary to remedy the identified procurement defects. The Federal Circuit ruled that the "narrowly targeted" standard violates and is contrary to the "highly deferential" "rational basis" standard under the Administrative Procedures Act ("APA") that the Federal Circuit has consistently used to review agency corrective action.
As stated by the Federal Circuit, "corrective action only requires a rational basis for its implementation," or, as otherwise stated, "whether the contracting agency provided a coherent and reasonable explanation of its exercise of discretion." Applying this to the instant facts, the Federal Circuit, in finding the agency's corrective action reasonable, stated that the agency "was not required to address every option, but rather to provide a reasonable corrective action and adequately explain its reasoning for doing so." The Federal Circuit further stated that "[e]ven if we agreed … that the [agency] had other, better options available," "we … refuse to 'substitute [our] judgment' for that of the [agency] by determining whether there was another, perhaps preferable solution." In contrast, "[a]sking whether a selected remedy is as narrowly targeted as possible to any identified error in the bidding process requires more than a finding of rationality or reasonableness."
The decision is Dell Federal Systems, L.P. v. United States, Fed. Cir. Nos. 2017-2516, 2017-2535 & 2017-2554 ("Dell"), publicly issued Oct. 5, 2018. The case involved an estimated $5 Billion procurement for multiple indefinite-delivery, indefinite-quantity ("IDIQ") contracts for "commercial-off-the shelf" (COTS) computer hardware, such as desktop computers, tablet computers and printers, over a 10-year period. The procurement was conducted on a lowest-price technically acceptable (LPTA) basis. 58 proposals were received, of which three were rejected as non-responsive. The agency awarded nine contracts, including one to Dell, after finding the other 46 unacceptable on various grounds, without holding discussions.
Twenty-one unsuccessful offerors protested to GAO. The agency conducted an internal review in response to these protests, and determined to take corrective action. The corrective action consisted of reopening the procurement, holding discussions with all responsive offerors, inviting and evaluating revised proposals and making new award decisions. The GAO dismissed the protests based on the stated intended corrective action. In order to level the playing field (since the awardees' prices had been disclosed in the debriefings and award notices), the agency decided to disclose all offerors' pricing.
Several of the original awardees then protested to the COFC. While the protestors did not dispute that there had been procurement errors, they challenged the corrective action as being overbroad and not narrowly targeted to the errors found. The COFC agreed. The COFC found that the agency had rationally identified two procurement defects relating to (1) "ambiguities in the Equipment Submission Form," and (2) the agency's failure to hold discussions." However, the COFC found that the agency's corrective action "is not rationally related to any procurement defects," and there was "a more narrowly targeted post-award solution that the [agency] entirely failed to consider" – namely, clarifications and reevaluation. (Emphasis added). The COFC therefore enjoined the agency's proposed corrective action. Both the Government and various intervenors appealed, challenging the COFC's "narrowly targeted" standard of review, as well as the COFC's unreasonableness decision.
As stated above, the Federal Circuit reversed and reinstated the agency's chosen corrective action. First, the Federal Circuit held that the COFC's "narrowly targeted" standard violated the Federal Circuit's precedent and APA standard of review. Second, the Federal Circuit held that the agency's proposed corrective action was reasonable and logically related to the identified procurement defects. The Court further held that the agency's pricing release was reasonable under the circumstances.
The bottom line is that the Federal Circuit's ruling means that agencies will have much greater leeway in formulating corrective action, so long as (1) there is a reasonable nexus between the procurement errors identified, and the chosen corrective action, (2) the agency rationally explains and justifies its choices. Conversely, it will be much harder for adversely-impacted offerors to challenge corrective action. Certainly, the "narrowly targeted" standard is now out. However, that does not mean that all corrective action challenges are foreclosed. The Federal Circuit did not depart from its prior decisions authorizing and sustaining corrective action challenges in appropriate cases. Agencies will still need a legally sustainable basis for taking corrective action, and will not be able to act arbitrarily. However, the review standard in such cases will be the traditional, highly deferential, APA review standard, and protestors will have a correspondingly higher hill to climb and heavier burden of proof.
Hopewell Darneille is responsible for the contents of this Article.
© 2018 Jackson Kelly PLLC
Hopewell Darneille
© 2023 Jackson Kelly PLLC. All Rights Reserved.


text:
Messy land ownership in Marawi complicates rehabilitation
May 15, 2018 8:46 AM PHT
Carmela Fonbuena
The government creates an adjudication board – the Land Dispute Arbitration Committee (LDAC) – to address concerns arising from the large-scale land titling program. There are many ways it could turn into a nightmare.
MANILA, Philippines – Norhata Bacarat's name – in red ink and bold letters – is written on a bullet-riddled wall of an abandoned store in Padian inside the former battle area in Marawi City
She is the "owner", according to the graffiti. A phone number accompanied the name, perhaps a plea to be contacted when there are questions about the property. But it was out of reach when Rappler called.
On May 10, the site of fierce clashes with local armed groups linked to Islamic State was again closed to the public after a month-long Kambisita, an activity the government arranged to allow residents to see their war-ravaged homes and collect remaining valuables before the structures are demolished.
There is fear sweeping among residents that they could lose their land when the government begins rehabilitation work.
The fear is rooted in the messy land ownership in the city. It's bad in the Philippines generally, but it's worse in Marawi City.
Many residents do not have official titles for the land they occupy while those who do have titles might find them overlapping with other claims. Government maps are not always reliable either.
There's paranoia that the situation will make it easy for the government to take the land for its plans to widen roads, make way for easements on both sides of Agus River, and other public infrastructure. (READ: Q and A: Marawi rehab chief seeks to ease fears of land grabbing)
Adjudication board
The messy land ownership in Marawi is going to complicate the rehabilitation work and the government knows it.
"It will be a problem. It will take time. It will be a lot of work. There will be frustrations for the losers and winners will be rewarded with a title later on," Assistant Secretary Felix Castro Jr, manager of the Task Force Bangon Marawi (TFBM) Field Office, told Rappler.
The government created the adjudication board – the Land Dispute Arbitration Committee (LDAC) – to address concerns that will arise from the large-scale land titling program.
In the absence of a title, Castro residents may present documents such as tax payments and papers from their respective barangay chairmen.
"It's an adjudication board to determine who really is the owner. Of course you will have to show your titles. If you do not have, you have to have proof that the land is yours. There are instances that there are many claimants and this has to be verified so that the real owner will be determined," said Castro.
Proof of ownership
There are many ways it can get complicated. At a consultation meeting, resident Drieza Lininding asked the government to also stop banks from taking mortgaged land in the battle area.
He said there are cases when relatives were able to mortgage the lots without informing the occupants.
"Our appeal is for the government to allow moratorium so that owners of affected lands or properties could settle their debt and made aware of this," Lininding said.
"Some do not know that their land or properties were already mortgaged by some unscrupulous people, some maybe their relatives, so we need to look into this before touching or taking anything inside MAA [the Most Affected Area]," he said.
Marawi City Mayor Majul Gandamra said the local government will be working closely with the task force of the national government although the members of the adjudication board have not been announced yet.
"It will probably be led by a lawyer with proven integrity and competence. The members will be coming from different sectors including civil society organizations," Gandamra told Rappler.
Warning from experts
Failure to manage land disputes could create new conflicts, warned a paper by planning experts Ica Fernandez, David Garcia, and Assad Baunto. (READ: Experts push for community-led Marawi rehabilitation)
"As in many post-conflict case, the option of strictly "no title, no return" policy may feed into extremist narratives of Christian dispossession of Muslim land, and therefore is not recommended," said the report published by The Asia Foundation (TAF).
The government should recognize traditional and cultural rights of residents, it said. "As with the rest of the region, many pre-colonial, traditional, and informal governance structures and practices around land, housing, property retain legitimacy alongside the laws of the present Philippine state."
The report said residents should be involved in resolving land disputes by tapping them, for example, to help map their own communities and correct and reconcile possible errors in official records.
The authors of the report put up a web site – www.openmarawi.com – where different maps and information are uploaded.
June groundbreaking
On May 10, the battle area was closed again to residents. The groundbreaking ceremony for the rehabilitation – which will begin with the demolition of structures – is scheduled in June.
The contract for the rehabilitation work has yet to be awarded. The design plans of Chinese-led Bangon Marawi Consortium, which government handpicked, will be subjected to a Swiss challenge.
But the experts also warned against starting demolition before resolving land disputes. "Debris clearing can only be done after the proper documentation of rights and ownership on housing, land, and property," according to the report published by TAF.
There might be a situation where the remaining walls may be the only evidence to mark where properties begin and end.
"Given the lack of comprehensive land records, it is important that these claims and their links to any existing physical features be documented before prematurely clearing and developing the most affected area," the report said.
Based on government's timeline, it will take about 18 months to reconstruct public infrastructure in the former city proper. The residents will be allowed back to rebuild their homes later.
For residents, a year-and-6-months is a long wait. But experts are concerned it might not be long enough to settle all land disputes in the former city proper.
The government was urged to manage expectations. – Rappler.com
Top photo: PROOF OF OWNERSHIP. Norhata Bacarat finds her property in rubbles when she returns to her home in Padian, Marawi City during the Kambisita on May 8, 2018. Photo by Manman Dejeto/Rappler


Still don't think KT is back in full training, Chris Davies said on Monday that he'd be at least a week before joining in with the group.
Hope I'm wrong as it would be great to have him back.
He's not kicked a ball for 3 months though so he won't be risked v Valencia.
Hope when Ntcham gets a chance he can show the kind of form we've seen in the past, coz he's got huge potential but lately he's fell right down the pecking order.
Still bickering about stawnin oan baws and thug goalies?
Someone said he would be ideal to fire balls through forward to Burke / Weah / etc. I think he can excel and save Brown for the tougher away games maybe.
I feel like he was getting a bit too comfortable, but with Brown, McGregor, Christie undroppable at the moment he's really gonna have to show his quality when he gets an opportunity.