The unconfined compressive strength test is the widely accepted protocol to investigate the strength properties of lime amended soil. As a quality control measure, specimens for unconfined compression test are prepared at a predetermined maximum dry density (MDD) using standard Proctor test. Replicating MDD in a small sized mould is difficult and subject to errors, which normally arise due to inconsistent compaction efforts. The conventional method for preparing specimens involves driving a core sampler into the compacted soil to extract the specimen. The approach proves to be laborious and is associated with high material usage, as such is not ideal for investigations of many variables. To address these challenges, an alternative protocol for specimen preparation at a controlled dry density was devised. In this study, a statistical analysis of the density values was used to validate the method. The regression analysis was employed to calibrate the compaction effort for a specified target density. The method offers manifold benefits such as;•Improved quality of specimens.•Reduced variability of UCS test data.•Efficiency.•Reduced material usage. Specifications table2 and class F flyash were used as stabilising agents. Sisal fibers were used as reinforcing elements at the content of 0.75%. The minimum lime demand for the soil was approximated by Eades-Grim test according to ASTMD 6276 [The soil was initially characterised in accordance with ASTM D1140-17 [TMD 6276 .The fibeThe mixture of the soil composite was allowed to mellow for 1hr according to  in a watA special hydraulic jack system was devised for compacting stabilised soil composite sample see a.The appThe mould size used herein was 55 mm diameter and 120 mm height see b, and waThe specimen extrusion set-up is shown in During compaction, deformation of the soil exerted hoop force F perpendicular to the interior surface of the mould. The hoop force F = P for equilibrium state after plastic deformation of the soil. The reactive force of the mould balanced the hoop force at the end of the compaction. At the incipient of specimen movement refer c, staticThe quality control aspects ensure that the fabricated specimens possess homogeneous properties. In respect of fiber-soil composites, uniformly distributed macrostructure and mass continuity are the fundamental parameters that dictate mechanical performance of the tested specimens. The variation of test results can be significantly minimised if great care is taken when preparing the specimens. Albeit, ASTM5102 procedure B stipulates that specimens for unconfined compressive strength test should be prepared by adapting ASTM D 698, extraction of specimens in the protocol is slow and labour intensive. Besides, material usage is high for experiments with several variables. In addition, human errors in counting number of blows (for 4.5 kg/2.5 kg rammers) lead to over compaction or under compaction of the sample. In particular, quite unlike standard compaction, this process cannot be automated.2) of the soil composite.In this study, variation in compaction effort for soil layers was controlled by load gauge. However, it was imperative to determine required load level in order to achieve target dry density as obtained from the preliminary standard Proctor compaction test. To effectively estimate compaction load, regression analysis between dry density and compaction pressure was employed. The trial specimens were prepared with compaction pressures of 1.5 MPa, 3 MPa, 5 MPa 7.5 MPa and 10 MPa at the constant OMC obtained from the preliminary standard Proctor test. The dry density of the specimens at specific compaction pressure was then computed. The compaction pressure and dry density were computed using Eqs. (3) and (4) respectively.2) shows that 99% of the data variability from 1700 kg/m3 to 2040 kg/m3can be well explained by the model.It can be seen that the required compaction pressure can be estimated by the model in The validation of the quality control method was carried out by utilising the regression model with known dry density obtained from standard Proctor test.The preliminary compaction test results of the soil-lime-flyash mixture are shown in 3.To reproduce this density under static compaction, the compaction effort was determined. Therefore, regression model  and (8) subject to Eq. (9).If coefficient of variation is taken as maximum acceptable deviation, then conditions in Eq. (10) and denoting CV = β give;,n,p).For β = 2.9%,1.9%, 0.9% and 0%,the 4 × 5 matrix could be generated with elements representing likelihood and probability of success of a given number of specimens (n,P(xThe above matrices imply that if 3 specimens are prepared, it is likely to get 2 or more density values within approximately ± 3% deviation from the target density. When the acceptable deviation range is reduced to approximately ± 2%, the minimum number of specimens should be 5.On the basis of material usage and time, 3 is the optimal number of specimens.The conventional method to test strength properties of lime stabilised soil is by conducting a series of unconfined compressive strength tests of the cured soil as stipulated by ASTM5102 .The dry •The method is more practical to replicate MDD in a small mould of diameter to height ratio of 1:2.5.•The compaction pressure of any given soil must be well calibrated in order to achieve desired density.•For every 3 specimens, at least 2 will fall within ± 3% of the target density•For a set of at least 3 specimens, it is very likely that 3 specimens will fall within ± 3% of the target density.•When acceptable density range is reduced to ± 2%, a minimum of 5 specimens is recommended.•On the basis of minimising material usage and time,3 specimens are recommended.The analysis of the proposed method indicates that;


Microbial communities are pivotal in the biodegradation of xenobiotics including pesticides. In the case of atrazine, multiple studies have shown that its degradation involved a consortia rather than a single species, but little is known about how interdependency between the species composing the consortium is set up. The Black Queen Hypothesis (BQH) formalized theoretically the conditions leading to the evolution of dependency between species: members of the community called ‘helpers’ provide publicly common goods obtained from the costly degradation of a compound, while others called ‘beneficiaries’ take advantage of the public goods, but lose access to the primary resource through adaptive degrading gene loss. Here, we test whether liquid media supplemented with the herbicide atrazine could support coexistence of bacterial species through BQH mechanisms. We observed the establishment of dependencies between species through atrazine degrading gene loss. Labour sharing between members of the consortium led to coexistence of multiple species on a single resource and improved atrazine degradation potential. Until now, pesticide degradation has not been approached from an evolutionary perspective under the BQH framework. We provide here an evolutionary explanation that might invite researchers to consider microbial consortia, rather than single isolated species, as an optimal strategy for isolation of xenobiotics degraders. Interactions of various natures between bacterial species that stabilize the community diversity can then arise and persist, but are most likely to disappear when the structure is disrupted4. In mixed liquid cultures, competition for a single resource is supposed to lead to competitive exclusion5, although very recent research has shown that cross-feeding may stabilize coexistence on a single carbon source6. If the breaking down of the resource includes both private and public goods, natural selection could favor the emergence of dependency between species, and therefore coexistence, through adaptive gene loss according to the Black Queen Hypothesis (BQH)8.Microorganisms in nature usually co-exist as communities whose complexity is under the influence of local environmental conditions and interactions between individuals. Spatially structured environments, such as biofilms, are more prone to support coexistence of multiple species consuming the same resource because access to the resource and its metabolic by-products is conditioned by the structure of the environmentet al. formulated three key characteristics for a function to follow the BQH. The function allowing consumption of the resource must be i) costly, ii) essential and iii) leaky. Also, Estrela et al.9 and Oliveira et al.10 recently specified the conditions that may favor the emergence of interdependency, which include low privatization and low cost of secretion. Atrazine is one of the most heavily applied herbicides worldwide which is relatively persistent and mobile in soil, and whose degradation products can be traced in soils decades after application12. Atrazine can be mineralized by microorganisms and is a source of nitrogen16. Its entire biodegradation pathway is known  involved in its mineralization are most often located on plasmids or on catabolic cassettes delimited by insertion sequences18. Because of the cost of maintaining plasmids, atrazine genes can be easily lost after a small number of generations in culture media without atrazine19. However, in nitrogen limited environments, atrazine degrading capacities must be conserved because mineralization of atrazine leads to the essential delivery of nitrogen20. Also, metabolites, such as desisopropylamine, aminoethanol and desethylamine, are produced and released in the environment during atrazine degradation21. Consequently, atrazine biodegradation is (i) conferred by genes costly to maintain, (ii) essential under N-limited conditions because it provides nitrogen supply, and (iii) benefits to other members of the community because metabolites leak in the environment. Atrazine biodegradation is therefore a good candidate function to test the BQH predictions, and in particular the emergence of dependency between species in a spatially unstructured environment.Morris Chelatobacter sp. SR3822, Pseudomonas sp. ADPe19, Arthrobacter sp. TES23 and Variovorax sp. 38R24 are all originally soil bacteria isolated from arable soils exposed to atrazine. They are all able to at least partly degrade either atrazine or its intermediary products . When evaluating the mineralization potential of the s-triazinic cycle, results are quite contrasted with a significant increase of the mineralization potential in the atrazine only supplemented medium , and significant decreases or no change in media supplemented with combinations of two or all three nitrogen sources. Altogether, these results indicate that the upper part of the atrazine degradation pathway  has been improved in the evolved consortia, presumably leading to increased intermediary metabolite production, such as aminoethanol, ethylamine or hypoxanthine21. While the upper pathway is constitutively expressed, studies have shown that the lower pathway was repressed by the presence of ammonium in the environment26. It was therefore quite likely that the stronger accumulation of intermediary metabolites and their consumption by consortium members led to an increased concentration of ammonium in the environment explaining at least partly the decreased mineralization potential of the s-triazinic cycle in evolved consortia compared to ancestors.We then evaluated the atrazine mineralization potential, using nts Fig.  and compTM assays. We found that only Arthrobacter sp. TES was able to grow in isolated culture, while the three other members of the evolved consortia depend on Arthrobacter sp. TES to grow in this medium  containing citrate as carbon source (5 mM) were supplemented with varied nitrogen sources combinations 2SO4, or 2 and 3 sources combinations) with a final nitrogen equimolarity of 1.4 mM (7 media). The evolution experiment was realized in 96-wells 2 mL Deepwell microplate at 28 °C without agitation during 23 growing cycles of 3.5 days each. For each new cycle, 35 µL of previous culture was used as inoculum for the next cycle into 700 µL fresh medium  x 23 cycles ~ 100 generations)). An aliquot of each cycle culture was mixed with glycerol  and preserved at −80 °C.Four strains with variable atrazine degrading abilities  containing the antibiotic for which it was to be resistant. Resistant growing colonies were collected and cultivated in their specific liquid MS medium to ensure that they have kept their metabolic ability towards atrazine. This procedure was repeated to confer a second resistance to each strain. Ten-fold successive dilutions of T1 and T23 “community” lines were done and inoculated onto LB agar plates supplemented with different antibiotic combinations. After 3-days of growth at 28 °C, CFU were counted across 3 dilution levels.atzA/atzD/trzD and atzC/trzN/atzB genes were used to determine the atrazine degradation repertoire of each strain of the ancestral and evolved “community” lines. 3 or 4 isolated colonies from the ancestral and evolved “community” lines were resuspended in water and served as DNA template for PCR reactions. PCR reactions were conducted using the following primers  or  at a final concentration of 10 µM each, the temperature of primers annealing being of 57 °C and the elongation time of 1 minute.Multiplex PCR targeting 600nm in 200 µL fresh medium supplemented with 4000 dpm final of 14C atrazine labelled either on the ethylamino chain or on the s-triazine cycle in triplicate. Cultures were then placed at 28 °C. A whatman® 3 mm Chr paper soaked with barite (saturated solution 0.37 M) was placed and sealed on the top of the 96-wells plates and replaced periodically during 3.5 days. Dried papers were fluorography printed on Storage PhosphorScreen (Molecular Dynamics®) for 2 days. Screens were scanned by a phosphorimager (Storm 860 Molecular Imager). Indirect reading of released radioactivity intensity was done by ImageQuant 5.2 software, and referred to a standard curve. Mineralisation percentages of lines were determinate with respect to initial radioactivity dosage realized on 200 µL of each culture medium with a Beckman® liquid scintillation counter. Significant differences between ancetral and evolved mineralization percentages were assessed using Tukey’s test (p < 0.05).Ancestors and “community” lines that have evolved on the four media containing atrazine were inoculated at ~0.001 ODTM. All lines were inoculated at ~0.001 OD600nm in 400 µL of their evolution medium (n = 3 for each replicate of each line) in Bioscreen TM plates. Plates were incubated 3.5 days at 28 °C and the evolution of OD600nm was monitiored every 10 minutes after a 10-seconds gentle shake. Population dynamics characteristics of each strain that has evolved in “community” lines was also measured the same way starting from 10 CFU isolated from the evolved “community” lines as inoculums and inoculated at ~0,001 OD600nm. Those data were used to determine the maximal OD600nm. Cultures that did not reach 0.1 OD600nm at the end of the 3.5 days were considered as non-growing. Significant differences between ancetral and evolved mineralization percentages were assessed using Tukey’s test (p < 0.05).The population dynamics of ancestral and evolved “community” lines were measured using Bioscreen600nm and final population densities, after a 3.5 days cycle, were measured for each strain by CFU plating on TY medium supplemented with the appropriate antibiotics combinations. Non-overlapping 95% confidence interval were considered as significant .For each strain, ten CFU isolated from each community-evolved lines (n = 3) in MS-Citrate Atrazine were used as inoculum pools. 4-species consortia, as well as all possible duos and trios were reconstructed and compared to monocultures. Mixtures and monocultures were inoculated at ~0,001 OD


APOE ε4 allele.This study describes a gauge for brain aging predictive of cognitive decline and Alzheimer's disease synergistic with the  APOE ε4 allele are the two biggest risk factors for Alzheimer’s disease (AD) and declining cognitive function. We describe a universal gauge to measure molecular brain age using transcriptome analysis of four human postmortem cohorts  free of neurological disease. In a fifth cohort of older subjects with or without neurological disease , we show that subjects with brains deviating in the older direction from what would be expected based on chronological age show an increase in AD, Parkinson’s disease, and cognitive decline. Strikingly, a younger molecular age  protects against AD even in the presence of APOE ε4. An established DNA methylation gauge for age correlates well with the transcriptome gauge for determination of molecular age and assigning deviations from the expected. Our results suggest that rapid brain aging and APOE ε4 are synergistic risk factors, and interventions that slow aging may substantially reduce risk of neurological disease and decline even in the presence of APOE ε4.Advanced age and the  How these risk factors relate to each other is an open question.Quality of life in old age is often compromised by dementia, mild cognitive impairment, and declining mobility. Dementia is common in the elderly with a prevalence that rises with age from about 5% in people 71–79 yr old to nearly 40% in people older than 90 yr in the United States . AlzheimStudies have characterized age-related differences in transcription in the human brain . These s5'—C—phosphate—G—3' (CpG) methylation sites has defined a subset of 300–500 sites whose level of methylation correlates very strongly with chronological age , APOE ε3 , and APOE ε4   . APOE ε3bout 76% . APOE ε4fourfold . There a alleles . APOE ε4athology . APOE ε4y bodies  and perhy bodies  with somAPOE ε4 status as risk factors for AD, focusing most heavily on LOAD and cognitive aging, for which we are most powered. Our findings suggest that brain aging is an important risk factor for AD and acts synergistically with APOE ε4 and may have important therapeutic implications for treating this and other late onset brain diseases.Here, we develop a reliable transcriptome-based gauge of molecular brain aging and use this tool to determine how rapidly brains have aged compared with the average in a cohort. This method correlates well with the DNA methylation-based clock . We use We used five human postmortem brain cohorts to develop and test a transcriptome-based biological brain age gauge. Characteristics of these cohorts are described in Subject-level cohort characteristics of the CM cohort.Table S1 Age-regulated genes used to create molecular ages.Table S2 P = 10 × 10−8). Older subjects may generally show a reduction in correlation due to a time-dependent divergence in differences in aging rates among individuals in the cohorts. It could also be due to a “survivor effect,” that is, the oldest subjects are more successful agers than their younger counterparts and have relatively younger brains.The CM cohort showed a high correlation of molecular age and chronological age at the time of death . We nextCharacteristics of the ROS-MAP cohort.Table S3 A proxy for the rates of brain aging was obtained as the difference between molecular age and chronological age for each brain, termed Δ age see . We, thuP < 2.2 × 10−16), as had been previously shown . We next wondered whether the transcriptional and methylation methods would reveal similar deviations from normal aging by comparing Δ age in these two cohorts as determined by both methods. Indeed, there was also a highly significant correlation  in BC and in ROS-MAP  between the two methods , Tauopathy (7 × 10−3), Huntington’s (P = 2 × 10−11), ALS (4 × 10−3), basal ganglia disorders (1 × 10−10), and other neurodegenerative disorders as well as to psychiatric disorders, including anxiety (P = 1 × 10−4), depression (2 × 10−4), bipolar disorder (1 × 10−3), and schizophrenia (2 × 10−7). More specific pathway categories for down-regulated genes include “glutamate signaling,” “dopamine feedback in cAMP signaling” , and “rho GTPases” , and “sirtuin signaling.” One exciting possibility is that defective sirtuin function contributes to mitochondrial and oxidative phosphorylation defects, which then impair neuronal function. However, we cannot discern whether mitochondrial dysfunction causes defects in neuronal function, neuronal functional defects cause mitochondrial defects, or the two are causally unlinked.Interestingly, the other three categories of down-regulated genes are “mitochondrial dysfunction,” “oxidative phosphorylation”  significantly associates with risk of clinical diagnosis of AD, with AD subjects showing significantly older molecular aging than nondisease controls , tremor (involuntary oscillation of limbs), gait , and bradykinesia  and withP = 5 × 10−5, The strongest association of positive Δ age was with global cognition slope , faster global cognitive decline (P = 1.8 × 10−5), episodic memory decline (P = 1.2 × 10−4), visual-spatial ability decline (P = 1.3 × 10−4), perceptual speed decline (P = 5 × 10−5), working memory decline (P = 0.02), greater PD signs (P = 0.01), more severe dementia grade (P = 7.6 × 10−0.05), tangles (P = 1.0 × 10−5), and Lewy body pathology (P = 0.005) (Table S5). With the exception of dementia grade (P = 0.006), these variables were not significantly associated with methylation Δ ages (Table S5). However, depression score (P = 0.03), amyloid (P = 0.05), Parkinsonian gait (P = 0.03), bradykinesia (0.05), and PD pathology (0.001) were associated with methylation Δ ages. These findings suggest that both methods are useful predictors, and that the transcriptional gauge may have a broader reach in associating with the risk of brain dysfunctional endpoints in aging.We used the subset of ROS-MAP subjects that had both methylation and transcriptional data available (n = 336) to directly compare the impact of transcriptional Δ ages to methylation Δ ages (Table S5). As with the 430 subjects shown above, we found that older transcriptional Δ ages continued to be significantly associated with Alzheimer’s diagnosis , neutral (−5 y to +5 y), or older (+1 SD from the mean or +5 molecular years), and combined subjects that were homozygous for APOE ε4 with those that were heterozygous because there were only four homozygous subjects. We found that Δ age and APOE ε4 are synergistic risk factors for AD  as a proxy for the rate of aging. These methods were developed by entraining the algorithm on one cohort of disease-free brains and applying them to four additional cohorts. Our method showed a highly significant correlation between molecular and chronological age for all cohorts. We then examined association of aging rates (Δ age) determined in nondisease brains with AD brains in the ROS-MAP cohort, which is highly enriched in AD, and made several important findings. First, a positive Δ age  associates with risk for AD with a high significance. Second,  infancy , suggest “clock”  correlatAPOEε4 positive  and Banner Sun Health Research Institute (BSHRI). The SMRI collection contains human postmortem brain samples from four brain collections: the Neuropathology Consortium, Array Collection, New Collection, and Depression Collection. These specimens were collected, with informed consent from next-of-kin, by participating medical examiners. Diagnoses of unaffected controls were based on structured interviews by a senior psychiatrist with family member(s) to rule out Axis I diagnoses. Exclusion criteria included the following: 1. significant structural brain pathology on postmortem examination by a qualified neuropathologist or by premortem imaging; 2. history of significant focal neurological signs premortem; 3. history of a CNS disease that could be expected to alter gene expression in a persistent way; 4. documented IQ < 70; 5. poor RNA quality; and 6. substance abuse within 1 yr of death or significant alcohol-related changes in the liver.PE BrainGVEX also contains human postmortem brain samples from the BSHRI Brain Donation Program . EligibiTotal RNA was isolated by organic extraction (SMRI) or miRNeasy Mini Kit (BSHRI). To pass QC to library generation, RNA must have concentration of ≥100 ng/uL assayed by Qubit 2.0 RNA BR Assay or Xpose, and RIN score ≥ 5.5 assayed by Agilent Bioanalyzer RNA 6000 Nano assay kit. All total RNA from both SMRI and BSHRI collections were processed into rRNA-depleted stranded libraries for sequencing on the Illumina HiSeq2000 using the TruSeq Stranded Total RNA Sample Prep Kit with Ribo Zero Gold HMR . Libraries are sequenced on Illumina’s HiSeq2000 on a high output flow cell for 100-bp PE sequencing. Libraries are three-plexed per lane to reach 40 M paired-end reads per library.Fastq files go through adapter removal using cutadapt, and then the resulting adapter-trimmed FASTQ files are checked for quality using FastQC. A subset of 10,000 reads is used to estimate insert mean size and SD for use with Tophat. Tophat is used to align trimmed reads to the GENCODE19 reference . Expression level are then calculated using HTSeq and Cufflinks with custom scripts used to summarize the proportion of reads assigned to each RNA type . Parameters that account for unstranded read orientation for polyA and stranded read orientation for Ribo-Zero libraries were used for Tophat and Cufflinks. FASTQ files are trimmed for adapter sequence and base quality using cutadapt, then subject to FastQC quality checks. Alignment is carried out with STAR in two-pass mode to the GENCODE19 reference genome. The resulting alignments are then sorted and merged (if there were multiple pairs for a given sample) using NovoSort.The BrainCloud cohort comprises brains that are free of neurological disease from medical history and pathological report. Neuropathological examination was performed in all cases by a board-certified neuropathologist. Brain sections through several cortical regions and the cerebellar vermis were examined microscopically, including the use of Bielschowsky’s silver stain. Cases with cerebrovascular disease (infarcts or hemorrhages), subdural hematoma, neuritic pathology, or other significant pathological features were excluded from further study. Cases with acute subarachnoid hemorrhages that were directly related to the immediate cause of death were not excluded.http://BrainCloud.jhmi.edu/.More details can be found here , but in phs000424.v6.p1. The GENCODE RPKM values were converted to ENSEMBL using BioMart (ENSEMBL version 73).The GTEx cohort comprises brains that are free of neurological disease from medical history and pathological report. More details can be found here , but in https://www.synapse.org/#!Synapse:syn3219045.The ROS-MAP cohorts are community based cohort studies of aging in which all participants are organ donors . We usedFurther normalization and quality control procedures were applied to each of the datasets. First, outlier values for each gene were removed from each dataset (SD > 4). In each cohort, there were several samples for which a large number of genes were outliers. After confirming that those samples did not correspond to the oldest and youngest subjects, they were removed. Overall, these samples had lower than average RIN scores. After removing those samples, the remaining outlier values were imputed with a K-nearest neighbor algorithm . To put A combination of observed and unobserved factors can have large effects on broad patterns of gene expression and are often controlled for in gene expression studies . In our To estimate molecular age, we built a model linking gene expression to age using the cohort with the broadest distribution of ages and the most control subjects, CM. We used elastic net regression, which has emerged as the standard for predicting age based on DNA methylation data . The modP < 0.001, linear model) associated with age in the cohort. This lead to a total of 1,263 genes that were included in building the model. In each direction, the highest correlation between predicted and actual age occurred at α = 0.01 (CM to PE R = 0.66). At α = 0.01, the λ that maximized this correlation between the two cohorts was 99. In total, 834 genes contributed to the model.There are two parameters in the elastic net regression, α and λ. We chose parameters based on how well a model trained with the CM cohort could predict age in the PE cohort. The genes used for the model were those that were strongly significantly .


Plasmodium species express variant antigens which may mediate immune escape in the vertebrate host. In Plasmodium falciparum, the rif gene family encodes variant antigens which are partly exposed on the infected red blood cell surface and may function as virulence factors. Not all rif genes are expressed at the same time and it is unclear what controls rif gene expression. In this work, we addressed global rif transcription using plasmid vectors with two drug resistance markers, one controlled by a rif 5′ upstream region and the second by a constitutively active promoter. After spontaneous integration into the genome of one construct, we observed that the resistance marker controlled by the rif 5′ upstream region was expressed dependent on the applied drug pressure. Then, the global transcription of rif genes in these transfectants was compared in the presence or absence of drugs. The relative transcript quantities of all rif loci did not change profoundly between strains grown with or without drug. We conclude that either there is no crosstalk between rif loci or that the elusive system of allelic exclusion of rif gene transcription is not controlled by their 5′ upstream region alone.All  Plasmodium species which cause malaria in humans still is a challenge for the public health predominantly in underdeveloped countries. Plasmodium falciparum alone is still responsible for 445000 deaths per year, mostly children under five years or pregnant women1. Part of the virulence exerted by Plasmodium is caused by the presence of variant antigens expressed on the surface of the host’s infected red blood cells. Members of the best-characterized family of variant antigens, the P. falciparum erythrocyte membrane protein 1 (PfEMP1), play a central role in immune evasion. PfEMP1 are encoded by approximately 50–60 different var genes2 which are highly recombinogenic4, possibly through specific three-dimensional DNA conformations near breakpoints5, and possess a modular structure6. In order to successfully evade immune mechanisms exerted by the human host, var gene transcription is tightly controlled in a way that normally only one or two var genes are expressed. A number of factors are involved in this control and these include not only sequences in the 5′ upstream regions of var genes8, including untranslated ORFs10, the pairing of var promoters and var introns11, but also specific DNA/chromatin binding factors12 and the involvement of several chromatin modifiers (reviewed in13). Recently, the participation of non-coding GC-rich RNAs in var transcription control was shown14. The current model of var transcription regulation also suggests a specific subnuclear site in which var transcription occurs and to which var loci translocate in order to be transcribed. However, the exact factors and dynamics which orchestrate this process and license one var locus for transcription while excluding all other var loci are largely unknown. Still more elusive is what determines that a var locus and its associated histone modifications switch from an active to a silent state or vice versa.The infection with one of the five Plasmodium species but also in murine species is the pir (Plasmodium interspersed repeat) gene family15, and the biological function of gene products from this family is not well understood. Recent results indicate that their encoded proteins may function at different points of the parasite-host interface16. If results from Plasmodium chabaudi can be extrapolated to all Plasmodium species, pir transcription seems to be reset during mosquito passage and reinfection17 and earlier evidence pointed to transcriptional diversity18 and quick switching in several models20. A recent study implied a specific PIR protein of P. falciparum  as a factor involved in the pathogenic process of erythrocyte rosetting22. Another study revealed that a specific motif in RIFINs promoted binding to leucocyte immunoglobulin-like receptor B1 (LILRB1) or leucocyte-associated immunoglobulin-like receptor 1, thereby inhibiting activation of B-cells and natural killer cells which express the LILRB1 receptor23. This turns evident that at least some RIFINs can be understood as virulence factors. Importantly, VIR proteins of Plasmodium vivax also appear to mediate cytoadherence and participate in pathogenic processes and probably immune evasion25. In P. falciparum, the vast majority of rif genes are localized adjacent to var genes, often in a tandem organization. In version 36 of PlasmoDB, there are 221 genes in the 3D7 strain genome which encode PIR proteins. Of these, 158 are full-length RIFINs, 27 are truncated or defect RIFINs, and the remaining are STEVOR (subtelomeric variant open reading frame) or truncated or defect STEVOR. RIFINs can be categorized into two major groups of rif genes and RIFINs: A and B. These groups differ by a short conserved 25mer peptide sequence in the first half of the protein which is present only in the 97 A-group RIFINs in the P. falciparum strain 3D7 genome26. While the A-group RIFINs seem to be exported to the infected red blood cell (IRBC) surface, B-group RIFINs are believed to remain associated with the vesicular network in the IRBC (Maurer’s clefts). It is still unclear what controls rif transcription, a recent study pointed to the transcription factor AP2-SP27 which somehow seems to influence transcription in blood stage P. falciparum28. While earlier studies indicated that rif and var gene transcription may be controlled by similar factors29, no clear-cut allelic exclusion mechanism could be detected in other studies which specifically addressed rif transcription or switching30. A drawback in the study from Howitt and colleagues29 was that the tested rif promoter controlled the unique drug resistance marker blasticidin deaminase in their construct, turning a considerable baseline activity of the promoter essential in order to obtain transfectant parasite lines. This did not permit a genuinely “switched-off” state of the rif 5′ ups-controlled transgene. In another study, the activity profile of a 5′-rif upstream region appeared more related to var genes31 and no significantly different regulation could be discerned upon activation. Also, no “crosstalk” – understood as the influence of the activity of one promoter on the activity of remaining promoters - between 5′ upstream regions such as occurs in var gene regulation was detectable. In contrast, in the study by Goel and colleagues, phenotypic selection procedures pointed to the expression of a single rif gene in parasites with PfEMP1-independent rosetting22, supporting the view that allelic exclusion and crosstalk may occur. In order to settle the question if there are allelic exclusion and crosstalk between rif 5′ ups regions, we used three different rif 5′ upstream regions in bicistronic transfection plasmids. Previously, this approach was successfully applied on var promoters32. After transfection, we divided transfected parasite lines and in one culture we selected the growth of parasites which actively transcribed a drug resistance marker (human dihydrofolate reductase). Afterwards, we compared rif transcripts between pyrimethamine-derivate sensitive and resistant lines by RNAseq.Another major variant gene family which is not only found in human or primate rif genes and the proteins they encode, RIFINs, is unclear. In order to test if rif genes are expressed in a similarly coordinated way as var genes33, we created bicistronic plasmids similar to those used by Voss7 or Witmer31 and colleagues. In these, one resistance marker gene was controlled by the constitutive plasmodial heat shock protein 86/90 promoter and the other by potentially inducible rif 5′ upstream sequences. We chose three rif upstream sequences (ups) based on previous observations using wild-type parasites of the 3D7 lineage where rif transcripts from these 5′ upstream regions were detected20. This assured that functional promoters were being used. The size (~1500 nt) of the inserted putative promoter sequence was chosen observing the distance to adjacent ORFs . When testing the copy number of the hDHFR gene, we observed that the transfectant line containing the PF3D7_0200700 5′ rif ups showed only one copy. In contrast, the hDHFR gene in a freshly transfected parasite line containing the Photinus luciferase encoding plasmid pCLH (a derivate of pDC1034 where the chloramphenicol acetyltransferase was changed for a luciferase coding sequence from pGL2 (Promega)) appeared in approximately three copies per cell , we conducted a Southern blot analysis using a digoxigenin-labeled bsd-gene fragment as a probe. The observed pattern indicated that recombination occurred at the hsp86/90 locus and not at the PF3D7_0200700 rif 5′ locus . In parasites that were not grown in the presence of WR99210, few hDHFR transcripts from the modified PF3D7_0200700 locus were detected (RPKM ~3), corroborating previous qPCR results. In contrast, higher RPKM values for hDHFR as the most abundantly detected rif transcripts were observed in parasites grown in the presence of WR99210. This indicates that the hDHFR-controlling rif promoter can be either active or inactive, pre-requisites of a variant gene promoter. Also, the RPKM values of the modified PF3D7_0200700 were in the same range as other simultaneously active rif promoters . When the relative rif transcript quantities from other loci were compared between parasites grown under WR99210 pressure or not, no profound changes were found. This can be interpreted that the activity of the modified PF3D7_0200700 rif promoter did not influence transcription from other loci. Such a result is in contrast with similar experiments using var promoters where the activity of a var 5′ ups led to a substantial silencing of the remaining genomic var loci7. When analyzing the RPKM values of all transcripts , we observed that only one rif B type transcript was detected in slightly elevated levels in both treated or untreated parasites, while several rif A-type transcripts were present  and Giardia lamblia36 to Plasmodium. Each parasite seems to have developed different molecular mechanisms to ensure that antigenic repertoires are not prematurely exhausted. In Giardia trophozoites, an RNAi-based mechanism enables the translation of a single variant surface antigen per parasite37. In Trypanosoma brucei, selective variant surface antigen (VSG) production is controlled at the transcriptional level and also through genetic recombination. Single vsg genes are transcribed by RNA Polymerase 1 at a specific subnuclear expression site38 and the activation of vsg expression sites is associated with specific chromatin modifications (reviewed in39). Var gene expression in P. falciparum is tightly controlled at the transcriptional level in ring-stage parasites40 and all but one or two sites remain silenced. Silencing of sites is associated with a number of specific chromatin modifications, mainly at histone H3 which - when trimethylated at lysine 9 - recruits heterochromatin protein 112, initially perceived as a landmark of silenced chromatin in different cell types41. A still not answered question is why not all var sites are silenced and the reason for this possibly lies in the concentration of ncRNAs transcribed from a number of GC rich regions present in the nucleus which somehow seem to coordinate gene counting14. Overproduction of members of these ncRNAs lead to the simultaneous transcription of var genes – an effect which is also observed when histone deacetylases SIR2A and SIR2B are repressed or deleted42. Here, we tried to interfere with the transcription of members of the rif gene family using an approach that was successfully applied to monitor var gene transcription. Interestingly, from three different constructs, only one presented a variable transcription activity, which may be considered as a pre-requisite of the corresponding RIFIN to function in antigenic variation. For the other two constructs, which also did not show integration at genomic sites, we found either a permanently activated or silenced status. Notably, all tested 5′-upstream regions have several, almost completely identical regions in the genome but only one of the rif genes controlled by these identical regions seemed transcriptionally active to the same degree as the transgene . This var locus – PF3D7_0400200 – is annotated as a pseudogene and consists only of exon 2. PlasmoDB predicts also the presence of the var intron, which itself is also a promoter. It is unclear if RNA from this site is involved in rif transcription regulation. Our data do not support co-regulation of rif and var genes. Of note, the material analyzed in our study may also not permit any conclusion about rif-var co-regulation, since RNAs from middle/late trophozoite stage parasites were employed. Normally, var transcription has ceased in trophozoites 20 h post reinvasion43. It must also be reinforced that the shown RNAseq results are unique for this specific experiment. It may be expected that the rif genes which appear transcribed in the current samples change over time due to switching and that in another experiment different, dominant rif transcripts are detected. Additionally, it is unclear if the observed rif transcripts occurred in the same parasites or if they originated from different parasites, concomitantly with the activated PF3D7_0200700 locus in all parasites. Also, it appears that the untreated sample was slightly advanced in the erythrocytic cycle since a number of schizont related genes such as MSPs showed more transcripts in the sample not treated with WR99210 compared to the treated sample . Given the ambiguous results for both rif 5′ ups PF3D7_0900500 and PF3D7_1300400 which are either not integrated or may have been rearranged during the transfection procedure, no further conclusions can be drawn why constructs containing these 5′ ups led to the observed results of permanent silencing or activity, respectively.Based on our data, it may be postulated the chromosomal context surrounding rif promoted hDHFR was partially lower than in the related study from Howitt29 or Witmer31 and colleagues. This may be due to the fact that we used low concentrations of blasticidin for selection. Howitt and colleagues used 2 or 10 µg/ml blasticidin and copy numbers at 2 µg/ml were in the same range or slightly higher than in our episomal constructs. Witmer and colleagues also used 2.5 µg/ml blasticidin but their plasmid backbones contained the TARE/rep20 element, known to improve plasmid segregation44. When we compared results of the hDHFR marker transcript in the transgenic PF3D7_0200700 parasite line growing or not in the presence of WR99210, we found smaller discrepancies between the “on” and “off” in qPCR than in RNAseq. The reason for this may lie in the differing range of linearity of both techniques, although this was not specifically tested for.Here, the copy number of plasmids encountered in parasite lines bearing most probably episomal forms of rif gene family, specifically of the RIFIN A subset, is most probably not controlled by a mechanism related to allelic exclusion as is valid for var genes and that almost 100% sequence identity in rif 5′ ups regions is not sufficient to predict promoter co-activation or silencing. However, not all rif genes are transcribed meaning that there still must be some kind of controlled activation and repression. Considering the results from Guizetti and colleagues14, it may be postulated that ncRNAs are also involved in the activation of rif loci, with the difference that these ncRNAs allow for activity from multiple loci – similar to what is seen for var genes when certain ncRNA from GC rich regions are overexpressed14. From the study of Guizetti and colleagues, it is not clear what influence the var relevant ncRNAs had on rif gene transcription although these authors speculated that a co-regulation could happen. Importantly, there are ncRNAs for most rif loci with yet elusive function45. A specific transcription machinery in the periphery of the nucleus was postulated for var genes48. To date, it is unclear if rif genes are also transcribed from the same machinery. This could be elucidated by RNA FISH or other adequate methods to monitor nuclear substructures, and the created parasite line may be useful for this kind of experiment. It also still remains elusive if the B subfamily of rif genes is differentially regulated, given the fact that we found only one B-type rif gene activated in our cultures.As a central result, we showed that transcription of the 2, 5% CO2, 5% O2 atmosphere or in candle jars as described earlier49. Human blood and plasma were obtained from the local blood bank and ethical clearance for using this blood for this research was granted by the Ethics Committee of the Institute of Biomedical Sciences at the University of São Paulo (No. 842/2016). The medium was changed daily or every two days when parasitemias were low (<0.5%). Parasitemias were checked by Giemsa-stained thin blood smears. For transfection, the protocol suggested by Hasenkamp et al.50 was used. Essentially, 150 µl of cytomix-washed fresh red blood cells were electroporated in a BioRad Gene Pulser at 310 V, 960 µF with 40 µg purified plasmid and later mixed with 2*107 plasmagel-purified51 schizont stage parasites. On day 2 after transfection, Blasticidin (Sigma) was added at 2.5 µg/ml to the medium. At day 6 after addition of the drug, no more viable parasites were visible and the medium was changed every two days until the appearance of parasites on days 17–25 post-transfection.Parasites (strain NF54) were cultured under biological level 2 conditions at 5% hematocrit in human B+ blood supplemented with 0.5% Albumax 1 (Invitrogen) or 10% human B+ plasma in RPMI and 0.23% sodium bicarbonate under a 90% N52 was inserted. The blasticidin-deaminase coding sequence was excised from pBMNL106P-PpLuciBlast53  and inserted into the vector. Then, the hsp86 terminator sequence was inserted from pPF86 via SalI and BamHI. In this plasmid, the hDHFR resistance cassette from pRESA-GFP-HA54 was inserted via EcoRI/EcoRV inserting in EcoRI/SmaI. This plasmid was used to exchange the Calmodulin promoter for a 5′ upstream region from three rif genes which were previously shown to be transcribed in wild-type 3D7 cultures20. The putative rif promoter regions were amplified using oligonucleotides (forward/reverse) ctcgagatataaatttgtaaaaaccatgtg/ggatccttaattgtgatacgtatattatttaatg , ctcgagtattatatttttatatataattattcgtg/ggatccatttaatgtgatacttatattattttatg , and ctcgagatgtaatatattattatgttaatattc/ggatcctattgtgatacgtatattattttatg . The amplified fragments were 1400 to 1500 nt and the identity of the sequences was confirmed by semiautomatic sequencing in an Applied Biosystems 7550 sequencer. An outline of the different rif 5′ upstream region containing plasmid is given in Fig. The bi-cistronic plasmid used here is based on the pTZ57 (Thermo/Fermentas) backbone, where a cassette containing the hsp86 promoter fragment (BglII-NcoI) from pPF8651 and subsequent Sorbitol lysis55 and then harvested after another reinvasion. harvested in ring, trophozoite or schizont stage forms. Harvested IRBC were treated with 0.1% Saponin for 10 min at RT and then pelleted at 12000 g/4 °C for 5 min, washed once in 1 ml PBS and then resuspended in a volume of 100 µl using TE. Afterwards, 1 ml Trizol (Invitrogen) was added and the sample was stored at −80 °C until use. Total RNA was prepared following the Trizol protocol provided by the manufacturer. Final total RNA was dissolved in 20 µl RNAse free water and stored at −80 °C until use.After outgrowth of parasites resistant to 2.5 µg/ml Blasticidin, parasites were split into two parallel cultures and cultivated in RPMI/Albumax supplemented with either 2.5 µg/ml Blasticidin/2.5 nM or 5 nM WR99210 or solely 2.5 µg/ml Blasticidin. After 4 reinvasion cycles, parasites were synchronized by plasmagel floatation56 using 5× Hotfire Pol SYBR Mix (Solis Biodyne Inc.) on an Eppendorf realplex2 thermocycler. In order to monitor the hDHFR transcription controlled by rif upstream regions, the following oligonucleotides were employed: forward 5′-ctggttctccattcctgagaag/reverse 5′-ttgtggaggttccttgagttct. These oligos were predicted using Primer3 online software57 using the same settings as used for the design of var oligos and the internal control oligonucleotide pair for the amplification of seryl tRNA ligase (PF3D7_0717700)58. Relative transcript quantities were then calculated by the 2−ΔCt method59 using the seryl tRNA ligase transcript as endogenous control. The performance of the hDHFR oligonucleotide pair does not differ from seryl t-RNA ligase specific oligos (data not shown). To test for integration of plasmids, genomic DNAs (gDNA) were prepared from parasite cultures using the Promega genomic DNA preparation kit and aliquots of the gDNAs were tested combining forward and reverse real-time PCR oligos for hDHFR (see above) and blasticidin deaminase . PCRs were performed using Thermo/Invitrogen Elongase with the following thermocycling program: 94 °C, 40 s; 54 °C, 40 s; 65 °C, 4 min, over 30 cycles.Total RNA was converted to cDNA using the previously published protocolP. falciparum genome available in the Ensemble Genome database (Release 26) and to generate gene reads count table used in the following pipelines. Two packages built in Bioconductor60 were used for further analysis: edgeR61 and limma62. The first was used to filter and normalize the data set, while linear modeling and empirical Bayes to assess differential expression were performed with the limma package. Finally, the RPKM values of trophozoite-derived cDNA from cultures which were treated with WR99210 or not were loaded in MS Access and filtered for product ID containing “rif”, and the corresponding RPKM values for each rif gene were plotted against their ID.Total RNA was isolated from the harvested trophozoite-stage parasites with Trizol reagent following the manufacturer’s instruction. The concentration of the isolated RNA was quantified using a Nanodrop device  and the integrity of the RNA was measured by a 2100 Bioanalyzer . Paired-end sequencing cDNA libraries were constructed from two samples (grown with or without 2.5 nM WR99210 for 6 reinvasions) using a TruSeq RNA Sample Preparation Kit v2 low sample (LS) protocol , based on the manufacturer’s instructions. RNAseq was conducted in an Illumina NextSeq500 sequencer following the recommendations of the provider using mid output flow cells and a total of 10 million reads per sample. CLC Genomics Workbench 7.01 platform was used to remove the adapter and assess reads quality from the raw reads. The same platform was used to subject reads to the reference PF3D7_0200700 parasites, using the Wizard® Genomic DNA Purification Kit (Promega). 5 μg of each gDNA and 25ηg of plasmid DNA (pDNA) from the pTZ57PF3D7_0200700 construct, were digested using SpHI (Thermo Fisher Scientific). The probe was amplified using standard PCR conditions with digoxigenin-dUTP from DIG High Prime DNA Labeling and Detection Starter Kit I (Roche Diagnostics), using the Blasticidin deaminase cassette from the pTZ57PF3D7_0200700 construct as a template. The oligonucleotide primers used were 5′- atgggaaaaacatttaacatttc-3′ and 5′-aacacaaaacaatctggtgcat-3′. The Southern procedure was performed following the protocol provided by the manufacturer of the labeling kit (Roche), using Hybond N membranes  and a hybridization temperature of 44 °C. All washings steps were performed at room temperature.Genomic DNA was isolated from the WT (NF54) and NF54::pTZ57Supplementary FiguresSupplementary Dataset 1


Interestingly, irx2a deficient embryos had reduced expression of an essential MCC gene ets variant 5a (etv5a), and were rescued by etv5a overexpression, supporting the conclusion that etv5a acts downstream of irx2a to control MCC ontogeny. Finally, we found that retinoic acid (RA) signaling affects the irx2a expression domain in renal progenitors, positioning irx2a downstream of RA. In sum, this work reveals new roles for irx2a during nephrogenesis, identifying irx2a as a crucial connection between RA signaling, segmentation, and the control of etv5a mediated MCC formation. Further investigation of the genetic players involved in these events will enhance our understanding of the molecular pathways that govern renal development, which can be used help create therapeutics to treat congenital and acquired kidney diseases.The genetic regulation of nephron patterning during kidney organogenesis remains poorly understood. Nephron tubules in zebrafish are composed of segment populations that have unique absorptive and secretory roles, as well as multiciliated cells (MCCs) that govern fluid flow. Here, we report that the transcription factor  Nephrons contain a blood filter, a segmented epithelial tubule, and collecting duct2. The blood filter disperses fluid across sieve comprised of a fenestrated network of capillaries that are situated within a specialized basement membrane that is opposed by podocytes with interdigitating cellular extensions3. Once inside the tubule, the renal filtrate passes by a series of specialized proximal and distal epithelial segment populations that are responsible for step-wise nutrient reabsorption and metabolite secretion. Fine-tuning of salt balance and final excretion occurs through the distal segments and subsequent collecting duct.The vertebrate kidney is an architecturally intricate organ composed of nephron functional units that cleanse the circulation of metabolic waste and maintain fluid homeostasis5. In part, this is due to the complicated nature of kidney organogenesis, which involves the coordinated creation of nephrons that each contains a multitude of differentiated cell types in precise arrangements. For example, in its final postnatal form, a single human kidney can contain hundreds of thousands to upwards of 1.5 million nephrons comprised of over 20 differentiated cell types6. Further, multiple kidney forms are made during development, which involves the successive formation and degradation of several nephron-based organs. In mammals, the adult metanephros is preceded by a transitory structure termed the mesonephros, which develops after the embryonic pronephros arises from the intermediate mesoderm (IM)7. In fish, the mesonephros is the adult kidney and a simple pronephros functions during embryonic stages7. Despite these differences, the fundamental genetic and molecular composition of nephrons is conserved across vertebrate species based on the common expression of transcription factors and solute transporters that define the various differentiated cell compartments9.While the composition and precise organization of nephron regions is essential for proper kidney physiology, the developmental processes that control formation of cell types are not yet fully understood11 and disease14 because of its rapid development and nephron conservation. During the initial stages of nephrogenesis, renal progenitors arise from bilateral stripes of IM precursors15 and have been characterized by dynamic spatiotemporal gene expression patterns that are modulated at the outset by retinoic acid (RA) signaling18. Following a mesenchymal-to-epithelial transition (MET) of the renal progenitors20, the zebrafish pronephros consists of two parallel nephrons that are connected caudally by a common collecting duct, and will later undergo morphogenesis events rostrally to form a shared blood filter apparatus22. The epithelial tubule of the nephron is subdivided into functional regions known as the proximal convoluted and straight tubule segments  and the distal early and late  segments, the latter where the associated corpuscle of Stannius (CS) gland arises16. Each of these segments is comprised of unique differentiated transporter cells that possess a single primary cilium, which is a microtubule-based projection that extends from the apical surface into the extracellular space. Additionally, a population of multiciliated cells (MCCs) is dispersed in the intermediate region of the nephron corresponding to the PST and parts of the adjacent PCT and DE segments24. Furthermore, all cilia within the zebrafish kidney are motile and participate in the movement of filtrate through the nephron tubule25. Interestingly, while MCCs are a major renal epithelial cell type in zebrafish, analogous MCCs in humans have only been documented in the fetal kidney27 and in case reports of kidney diseases such as hypercalcemia, congenital nephrosis and glomerulonephritis34. These findings suggest that understanding the mechanisms that guide MCC formation may provide insights about kidney development and disease.In recent years, the zebrafish pronephros has emerged as a powerful model of vertebrate renal ontogenymds1/evi1 complex (mecom) transcription factor35. mecom subsequently promotes Notch signaling which negatively modulates a binary transporter cell versus MCC fate choice35. In part, Notch signaling inhibits expression of the etv5a transcription factor, while RA promotes etv5a to stimulate MCC development36. Moreover, etv5a and etv4 were found to have redundant functions since their combined deficiency caused a significantly greater decrease in MCC number than the knockdown of either factor alone36. These studies indicate that the MCC developmental pathway must be highly regulated, as the loss of any single factor did not completely impair MCC formation36. Consequently, the exact mechanisms that direct the specification of MCC remains are not yet completely elucidated.A growing list of genes and signaling pathways have been identified as participants in the networks that direct segmentation and MCC development during zebrafish renal organogenesis. RA both controls proximo-distal segment pattern and promotes MCC development by inhibiting expression of the iroquois (iro/Irx) genes encode transcription factors that belong to the TALE superclass of homeodomain proteins and regulate the patterning of tissue territories during embryogenesis in invertebrates and vertebrates, respectively38. Several Irx genes are expressed during kidney development in amphibians, mammals and zebrafish43. For example, irx1/2/3 transcripts are localized to the intermediate tubule region of the Xenopus pronephros41, and in the mouse metanephros are located in the intermediate region in the S-shaped body of developing nephrons and later to the middle section of the nephron, known as the Loop of Henle45. Knockdown of Irx2 did not alter pronephros tubule development in the frog embryo, however, leading to the hypothesis that it shares redundant activities with Irx1 and/or Irx341. Interestingly, the zebrafish homologs, irx2a and irx3b, are expressed in the central region of the pronephros tubule, in a domain which includes the PST and DE segments along with MCCs40. irx3b is required for DE specification, where the loss of this transcription factor results in the abrogation of slc12a1+ tubule cells, expanded proximal segments, and an expanded CS lineage46. In contrast, the function(s) of irx2a during pronephros development have remained undefined until now.The irx2a in PST and DL segment development as well as MCC formation in the zebrafish pronephros. We found that irx2a regulates expression of etv5a in part to control MCC fate choice, and that this regulation occurs downstream of RA signaling. These findings provide the first account that irx2a coordinates nephron segmentation and MCC development, which has implications for understanding kidney organogenesis across vertebrates.Here, we report novel roles for 16  on wild-type zebrafish embryos between the 5–28 ss to assess its spatiotemporal expression domain in the emerging kidney. irx2a transcripts were detected first at the 15 ss in a pattern consistent with the central region of the developing pronephros tubule, where irx2a transcripts continued to be expressed at the 20–22 and 28 ss  experiments to further define the occupancy of irx2a transcripts within renal progenitors. Prior studies have shown that cadherin17 (cdh17) expression demarcates the nephron tubule at the 28 ss47, while outer dense fiber of sperm tails 3B (odf3b) demarcates MCCs23. We found that irx2a transcripts localized both to cdh17+ and odf3b+ nephron cells  and forms a series of cell types which will comprise the tubule, and others that later contribute to formation of the blood filter16 Fig. . The epi 24 Fig. . Further 24 Fig. . To furt ss Fig. . Next, wlls Fig. , confirmpts Fig. . Interesers Fig. . As the irx2a during kidney development, we conducted knockdown studies by utilizing a morpholino (MO) that specifically targeted the splicing boundary between intron 1–2 and exon 2 of the irx2a pre-mRNA sequence48 , trpm7 (PST), slc12a1 (DE), and slc12a3 (DL)16. irx2a deficient embryos formed PCT and DE segments that were indistinguishable in absolute length from wild-type controls . Expression of irx2a was sufficient to rescue both PST and DL development in irx2a deficient embryos, which was found to be statistically significant based on measurement of absolute segment lengths  necessary to support pronephros development. Thus, the normal appearance of renal MCCs and segments in irx2asa10776 mutants is not likely due to genetic compensation by other factors.To explore the latter, we performed site-directed mutagenesis on our RNA Fig. . Given tirx2a and RA signaling to explore how irx2a further fits within the genetic cascades that regulate pronephros segment and MCC development. RA is a diffusible morphogen that is necessary to establish rostrocaudal patterning among pronephros progenitors during the earliest stages of nephrogenesis, and subsequently modulates the expression of many transcription factors to direct segment formation17. Furthermore, treatment with exogenous all-trans RA results in an expansion of proximal segment identities including MCCs at the expense of the distal segment regions35. Conversely, the inhibition of RA signaling by blocking its biosynthesis with the inhibitor N,N-diethylaminobenzaldehyde (DEAB) causes distal fates to be favored, while decreasing proximal lineages and abrogating MCCs35.We next sought to determine the relationship between irx2a expression within the pronephros. Wild-type embryos were treated with RA or DEAB at 60% epiboly and fixed at the 28 ss for WISH analysis. We found that treatment of wild-type embryos with exogenous all-trans RA resulted in a domain expansion and a distal shift of irx2a expression  observed in prostaglandin signaling gain of function studies66, and mecom loss of function studies35. Moreover, the tbx2a/2b transcription factors are requisite for DL development67. In the zebrafish kidney, expression of four other irx genes has been documented as well , denoting these genes as potential regulators of these segments68. Determining the relationship of irx2a with these various irx family members, and exploring the hypothesis that functional redundancy may exist between these related genes in renal progenitors, will further our understanding of the networks that underlie pronephros segmentation.Further, the relationship between Irx2a and the transcription factor genes Xenopus pronephros where Irx2 knockdown does not cause renal anomalies42, homozygous Irx2 mutant mice are healthy and lack obvious organ deficiencies69. Here, lacZ expression faithfully recapitulated the endogenous Irx2 domain in various tissues including the central nervous system, kidneys, pancreas, and lungs in Irx2LacZ/+ mice where the null allele was generated by targeted introduction of an IRES-lacZ reporter and neomycin resistance gene69. The intercross of these Irx2LacZ/+ mice produced progeny according to normal Mendelian ratios, and analysis of homozygous Irx2 mutants showed that the Irx2 transcript level was markedly reduced but did not result in protein production, signifying loss of function69. One explanation for the normal renal formation and function in Irx2 knockout mice may be functional redundancy with other Irx family members. Indeed, Irx1/2 were placed downstream of HNF1B during intermediate tubule development in the murine metanephros45. It is possible that evolutionary differences exist between Irx gene functions in zebrafish and other vertebrates including mammals70. Most vertebrates have six Irx genes that are organized into two clusters, IrxA and IrxB, arising from a duplication event that occurred early in their evolution71. Following teleost divergence from the tetrapod lineage, zebrafish underwent another whole genome duplication, which gave rise to four irx clusters and a single isolated gene locus. Of these, irx2b was deleted from the zebrafish genome77. Consequently, it is possible that neofunctionalization occurred where irx2a accrued adaptive mutations allowing it to perform unique functions during cell specification rather than dependently operating under the control of irx3b as the findings in the Xenopus studies predict41.In addition to the contrast between our findings and the Irx transcription factor has essential functions in MCC development. MCCs have motile cilia and are responsible for stimulating fluid propulsion across tissues in a synchronized and directional manner79. In these MCCs, each cilium is characterized by a bundle of microtubules organized in a 9 + 2 configuration that is surrounded by a plasma membrane and anchored to the surface of the cell by a basal body79. The motility of a cilium is then stimulated by dynein arms located in between the nine microtubule doublets, resulting in a bending movement due to the action of these motor proteins79. In contrast, most cells in vertebrates contain a single, non-motile primary cilium that predominantly functions in signal transduction79. Despite the known significance of MCC function during development and homeostasis, the anatomical location of these intriguing cells in mammals throughout the brain ventricles and airways, for instance, has historically precluded them from comprehensive genetic examinations. In recent years, however, the application of functional genomics has led to numerous advancements due to the use of various animal and cell culture models80. Efforts to better define the transcriptional programs and ancillary modulating factors of MCC differentiation have established Notch signaling as an essential regulator of multiciliated fate choice82. This mechanism was found to be conserved between MCCs located in the zebrafish embryonic kidney, murine respiratory tract, and Xenopus epidermis, where the latter has also served as an excellent model for the live imaging of MCCs85. Furthermore, the microRNA, miR-499, was recently discovered to control the expression levels of Notch and Delta-like1, thereby contributing another layer of control to the pathways that influence MCC differentiation86. Additional studies in Xenopus and mice lead to the identification of a Notch target, Multicilin, which is a small coiled-coil protein that acts in a complex with the E2f4/5 transcription factors to regulate multiciliogenesis through the activation of key centriole replication genes88. Other research in these model organisms have identified that members of the Rfx family89, including Rfx2/3, C-Myb, and FoxJ1, operate in evolutionarily conserved functions downstream of Multicilin to promote the genesis of motile cilia91.The current work implicates for the first time that an irx2a in MCC development  was conducted as described102. Anti-sense RNA probes were digoxigenin-labeled  or fluorescein-labeled , and generated by in vitro transcription using plasmid or PCR templates as described103. Embryos were mounted in glycerol and images were taken using a Nikon Eclipse Ni with a DS-Fi2 camera. Whole mount fluorescent in situ hybridization (FISH) was performed as described103 with the digoxigenin and fluorescein-labeled RNA probes used in WISH. Stains were developed with the TSA plus Cy3 and TSA plus Fluorescein kits (Perkin Elmer). Embryos were mounted in Poly Aqua-mount as described104 and imaged on a Nikon C2 confocal microscope. Z-stacks were processed with FIJI into max image projections, and all figures were assembled using Adobe Photoshop CS5.Whole mount irx2a splicingwith the following sequence48: 5′-ACGGAGAGCCCTTCAAAAATAAC-3′ (ZFIN Annotation MO2-irx2a) at 133 µM. The antisense standard control MO was used at was: 5′-CCTCTTACCTCAGTTACAATTTATA-3′ at 133 µM. MOs were synthesized and purified by Gene Tools, LLC , and resolubilized with DNase/RNase free water for storage at −20 °C. irx2asa10716 were obtained from the Zebrafish International Resource Center49. For genotyping irx2asa10716, we developed a restriction fragment length polymorphism assay. Genomic DNA was isolated from individual adult fins or embryos as described105. PCR amplification was performed with primers flanking the mutation site: 5′-GACCTTGTTTGCGATTCTGGAGCTGAAATC-3′ and 5′- GGGGTCCGAAGTGGCGATCTCTGCTAATGA-3′. The cycling conditions were as follows: initial 4 minute denaturation step at 94 °C followed by 45 cycles of 94 °C for 30 seconds, annealing for 30 seconds at 68 °C, 72 °C for 1 minute and a final extension step at 72 °C for 10 minutes. PCR products were purified using a Qiagen PCR Purification Kit and sent to the University of Notre Dame Genomics Core for sequencing. Restriction digest of the purified PCR product was performed using the enzyme Bts1Mut1 (NEB). The digest reaction  was incubated at 55 °C overnight. 10 μL of the digest product was then run on a 2% agarose gel.An antisense morpholino (MO) was used to block irx2a exons 2 and 3: forward 5′-GACGAAGACGAAGATGACGGAGATG-3′, reverse 5′-CTCGCATTTGTCCTGGATTTCAGCT-3′. Products were isolated by agarose gel extraction (Qiagen QIAquick Gel Extraction) and sequenced.20–30 uninjected and injected embryos at the 28 ss were homogenized in 500 μL TRIZOL (Ambion) and RNA was isolated according to manufacturer insructions. PCR amplification was performed using the SuperScript® IV First-Strand Synthesis System (Invitrogen) according to manufacturer instructions with primers to amplify the region between irx2a open reading frame (ORF) was PCR amplified using high fidelity TAQ polymerase from the Expand PCR kit (Roche) in combination with a PCR Mix solution , 4 M KCl, 1% Gelatin, 100 mg/mL BSA, and sterile H2O) and primers specific for irx2a: forward, 5′-ATTCGAATTCGCCGCCACC atgtcctatcctcagggttacctctaccagcccccgggctc-3′ and reverse, 5′-CGACCTCGAG ttaactcgacaggtaagattgggatcttactgtgaaaacctcgctggggt-3′. The forward primers were designed with a 4 bp anchor (bold print) followed by the 6 bp EcoR1 sequence , the Kozak consensus (underlined), and finally a sequence beginning at the start site (lowercase). Alternatively, the reverse primer contains the stop sequence (lowercase), 6 bp Xho1 sequence , and a 4 bp anchor (bold print). Amplified ORF was ligated into the pCS2 vector and transformed into DH5α competent cells (Invitrogen). Mutagenesis was performed on the irx2a.pCS2 construct to create irx2asa10776.pCS2 using the QuikChange Site Directed Mutagenesis Kit  as previously described106. The mutagenesis primers were forward, 5′-GGTGACGCGCCTCACTGCCTTTCCTCC-3′ and reverse, 5′-GGAGGAAAGGCAGTGAGGCGCGTCACC-3′. etv5a was synthesized from a previously reported rescue construct template33. All capped RNA (cRNA) was synthesized in vitro using the mMESSAGE mMACHINE SP6 Transcription kit (Ambion) and stored at −80 °C. Overexpression experiments were performed by injecting 20 pg of irx2a cRNA into wild-type embryos. For irx2a and irx2asa10776 rescue experiments, 20 pg of cRNA was co-injected with 133 µM irx2a MO. For etv5a rescue experiments, a combination of 220 pg etv5a cRNA with 133 µM irx2a MO was injected into wild-type embryos. For all rescue studies, replicate group sizes were a minimum of 30 embryos, and typically ranged between 40–60 embryos for each cohort.The zebrafish odf3b+ cells in both nephrons93. Each experiments was completed in biological triplicate, with group sizes of at least n = 10 embryos, and confocal imaging was performed with group sizes of a least n = 3 representative embryos. ANOVA and student t-tests were used to assess statistical significance. Graphs were created using GraphPad Prism software.To measure domain length in the pronephros, embryos were mounted laterally in glycerol on a bridge slide. Either the polyline tool on the Nikon imaging software was used to trace the expression domain, or the segment line tool in FIJI. MCC cell number was assessed by viewing embryos dorsally at the highest magnification on a Nikon SMZ1000 stereomicroscope, and counting the individual 16. For all chemical treatments, wild-type embryos were incubated and protected from light in vehicle control, 1 × 10−7 M RA/DMSO or 1.6 × 10−5 M DEAB/DMSO made with E3 from 60% epiboly to 24 hpf. The chemical was then washed off and the embryos were fixed in 4% PFA. These chemical treatments were fully penetrant and produced consistent results over three replicates with n = 40–60 embryos per replicate analyzed for these studies.all-trans RA and DEAB (Sigma-Aldrich) were dissolved in 100% DMSO to make 1 M stock solutions, as previously describedSupplementary Information


Multiple system atrophy (MSA) is a unique and fatal α-synucleinopathy associated with oligodendroglial inclusions and secondary neurodegeneration affecting striatum, substantia nigra, pons, and cerebellum. The pathogenesis remains elusive; however, there is emerging evidence suggesting a prominent role of neuroinflammation. Here, we critically review the relationship between αS and microglial activation depending on its aggregation state and its role in neuroinflammation to explore the potential of TNFα inhibitors as a treatment strategy for MSA and other neurodegenerative diseases. Multiple system atrophy (MSA) is a rare, rapidly progressing and fatal neurodegenerative disease of unknown etiology that is clinically characterized by a variable combination of parkinsonism, cerebellar impairment, and autonomic and motor dysfunction , 2. TogeTNFα, one of the key regulators in inflammation, belongs to the TNF ligand superfamily and is synthesized as a type II integral membrane protein occurring in a vast number of cell types. Within the central nervous system (CNS), microglia, astrocytes, and neurons are capable of synthesizing TNFα; however, activated microglia represent the main production site during neuroinflammatory processes , 35. FolNeuroinflammation characterized by microglial activation with secretion of many pro-inflammatory cytokines, in particular IL-1β and TNFα, has been implicated as main effector of the functional consequence of neurotoxicity, resulting in mitochondrial dysfunction , therebyThe importance of TNFα in the processes of development of PD is strengthened by animal models of PD, where elevated levels of TNFα are seen in a manner consistent with that of clinical PD , 61, 62.Due to the possible cyclic nature of αS aggregation/release and gliosis in MSA, interventions that target neuroinflammation may have the potency to slow the progression of the disease and increase the quality of life . Recent Therapeutic regimes that interfere with either the synthesis of TNFα or the downregulation of TNFα/receptor interactions may have a considerable benefit to patients with such conditions. The use of agents similar to thalidomide and its analogs will inhibit TNFα protein biosynthesis, which will circumvent classical receptor subtype agonistic effects on signaling pathways. Furthermore, thalidomide will display biological activity in the CNS as it is a relatively small molecule and can cross the blood-brain barrier (BBB). Thus, it offers a better strategy for treating TNFα overproduction-induced disorders than that of currently available large proteins that bind and lower soluble TNFα . Agents Thalidomide is widely known for its deleterious side effects since the late 50s/early 60s when birth defects were observed after pregnant women were prescribed thalidomide to treat morning sickness . In addiInhibition of the biological effects of sTNFα by etanercept and infliximab displayed beneficial properties against rheumatoid arthritis and other peripheral inflammatory diseases. Unfortunately, these agents are large macromolecules that minimally pass the BBB and thus will preclude their utility in CNS neurodegenerative disorders. BMS-561392 reduces the amount of sTNFα by inhibiting TACE; however, clinical phase II trials for rheumatoid arthritis have been halted due to mild hepatotoxicity , which mA recent study investigated the therapeutic efficacy of combining an unconventional anti-inflammatory therapy (lenalidomide, a small thalidomide derivative with immunomodulatory activity and therapeutic effects in multiple myeloma –93, withTNFα-dependent neuroinflammation may play a key role in MSA pathogenesis, and its relevance has been underlined in various models of synucleinopathy. Targeting TNFα with readily available drugs may constitute a promising disease-modifying treatment in this hitherto incurable disease.