COMT) single nucleotide polymorphisms (SNPs) with increased cisplatin-induced nephrotoxicity. In total, 551 patients were genotyped for the 1947 G>A , c.615 + 310 C>T (rs4646316), and c.616–367 C>T (rs9332377) polymorphisms. Associations between these variants and AKI grade ≥3 were studied. The presence of a homozygous variant of c.616-367C>T was associated with a decreased occurrence of AKI grade 3 toxicity  0.201, 95% confidence interval (CI) (0.047–0.861)). However, we could not exclude the role of dehydration as a potential cause of AKI in 25 of the 27 patients with AKI grade 3, which potentially affected the results substantially. As a result of the low incidence of AKI grade 3 in this dataset, the lack of patients with a COMT variant, and the high number of patients with dehydration, the association between COMT variants and AKI does not seem clinically relevant.Cisplatin is a chemotherapeutic agent widely used for multiple indications. Unfortunately, in a substantial set of patients treated with cisplatin, dose-limiting acute kidney injury (AKI) occurs. Here, we assessed the association of 3 catechol-O-methyltransferase ( Cisplatin is a widely used cytostatic agent that interferes with DNA replication by binding within (and between) DNA strands resulting in the inhibition of protein synthesis and DNA replication. It is approved for the treatment of many types of cancer such as testicular seminoma, non-small cell lung cancer, and head and neck cancer . Common COMT, were genotyped in a young woman who developed severe irreversible cisplatin-induced nephropathy. Both COMT polymorphisms were already known to be associated with cisplatin induced ototoxicity . AK. AK13]. Of patients with AKI grade 3 or higher, the patient files were studied for potential confounders, such as dehydration and renal and post-renal pathology and recovery. When the plasma creatinine concentrations of these patients did not return to baseline +20 μmol/L in 2 years after cisplatin administration, this was classified as irreversible cisplatin-induced nephrotoxicity.COMT SNPs were genotyped using predesigned Drug Metabolism Enzymes TaqMan® allelic discrimination assays on the Life Technologies TaqMan®7500 system . Assay IDs are listed in ™ and FAM™ fluorescent dyes. Using these assays, TaqMan®GTXpress Master Mix and 20 μg genomic DNA, the polymerase chain reactions were performed in a reaction volume of 12 μL. The thermal profile of the TaqMan® qPCR reaction consisted of 40 cycles of denaturation (95 °C for 20 s), annealing (92 °C for 3 s), and extension (60 °C for 30 s). Genotypes were determined by measuring allele-specific fluorescence using the TaqMan®7500 software v2.3 for allelic discrimination.DNA was isolated using the MagNA Pure 96 Instrument , from 500 µL of the whole-blood specimens using the MagNA Pure 96 DNA and Viral Large Volume Kit . The selected Distribution of genotypes was tested for Hardy Weinberg equilibrium; if p < 0.05, the genotype was excluded from analysis. The minor allele frequency (MAF) was also determined and compared to the MAF in the literature .COMT 616-367C>T and 1947 G>A were tested in the dominant model [COMT 615+310 C>T was tested using the recessive model [Associations of the SNPs with the endpoints were tested in the dominant model; heterozygous variant (HT) and homozygous variant (HM) versus wildtype (WT), or the recessive model; and HM versus HT and WT. Prior research was reviewed to determine if the variant affected the SNP in a recessive or dominant manner. nt model . COMT 61COMT polymorphisms p < 0.1 were considered for multivariable logistic regression analysis together with baseline variables such as age, gender, and cisplatin dosage, if possible. When Fishers’ exact test was used, no multivariable analysis was performed due to the large influence of chance on the outcome.Univariable associations of AKI with A total of 551 patients, with different malignancies, were included in this study. All genotypes were in Hardy–Weinberg equilibrium . BaselinCOMT c.616-367C>T was significantly associated with less AKI grade 3 when performing a dominant analysis  0.201 95% confidence interval (CI) (0.047–0.861)). Age ≥65 years was univariably associated with the incidence of AKI grade 3 ) but dosage ≥80 mg/m2 and gender were not  recovery after rehydration. Data on possible confounders such as fluid intake, co-medication, diarrhea, and nausea were not available and, due to the small sample size, could not be adjusted for in a multivariate analysis. However, these factors as a possible cause of AKI must be taken into account during cisplatin treatment. Other causes of AKI grade 3 have not been investigated regularly and urinalysis was performed only in a minority of patients. Moreover, there was large heterogeneity in the interval between blood draws, since these were only regularly scheduled in advance of each chemotherapy cycle and additional blood draws were executed based on clinical judgement of symptoms and hydration status. Therefore, temporary and asymptomatic elevations in plasma creatinine might have been missed in this study. Cisplatin-induced nephrotoxicity is currently mostly prevented with hyperhydration, magnesium supplementation or mannitol-induced forced diuresis, whereas none of these measures will guarantee that kidney function will remain stable . AmifostCOMT c.616-367 C>T is significantly protectively associated with the incidence of AKI grade 3 during cisplatin treatment. Despite the considerable size of this cohort, there was insufficient power and lack of data on hydration state to investigate the extent in which possible confounders such as dehydration affected this result. This study did not provide a rationale for pre-emptive genotyping of COMT SNPs to predict AKI in patients treated with cisplatin. The retrospective nature of this study necessitates prospective validation.In conclusion, we found that


Mosquito-borne diseases involving arboviruses represent expanding threats to sub-Saharan Africa imposing as considerable burden to human and veterinary public health. In Mozambique over one hundred species of potential arbovirus mosquito vectors have been identified, although their precise role in maintaining such viruses in circulation in the country remains to be elucidated. The aim of this study was to screen for the presence of flaviviruses, alphaviruses and bunyaviruses in mosquitoes from different regions of Mozambique.NS5 sequences, frequently detected in Mansonia seemed to correspond to defective viral sequences, present as viral DNA forms. Furthermore, three lineages of putative members of the Phenuiviridae family were also detected, two of which apparently corresponding to novel viral genetic lineages.Our survey analyzed 14,519 mosquitoes, and the results obtained revealed genetically distinct insect-specific flaviviruses, detected in multiple species of mosquitoes from different genera. In addition, smaller flavivirus-like This study reports for the first-time novel insect-specific flaviviruses and novel phenuiviruses, as well as frequent flavivirus-like viral DNA forms in several widely known vector species. This unique work represents recent investigation of virus screening conducted in mosquitoes from Mozambique and an important contribution to inform the establishment of a vector control program for arbovirus in the country and in the region. Vector-borne diseases caused by arboviruses such as the Rift Valley fever, dengue, chikungunya, Zika, or West Nile viruses , represent emerging and expanding threats in sub-Saharan Africa, and remain a major burden to global health, despite increasing funding allocated for their control and eradication . Every ytaxa or infrequently visited biotopes, and revealing novel or divergent viral groups  in the cDNA extracts prepared from 45/351 pools (12.8%). These results indicated the presence of flavivirus genome in 9 different species of mosquitoes from possibly 4 genera  was obtained from a pool of mosquitoes identified as Culex (Cux) antennatus (pool Moz 182). However, the association of an apparently Culex-derived viral sequence with this group was considered debatable given its high similarity with the viral sequences amplified from Mansonia (see discussion). The above mentioned lineages of cISF include the Cuacua virus, previously identified in Mansonia sp. [Ma. africana and Ma. uniformis.To further extend the characterization of the viral sequences obtained, a phylogenetic analysis was carried out using different methods. In all cases, the obtained phylogenetic trees indicated that none of the analyzed sequences had been amplified from bona fide arboviruses. Indeed, this is clearly revealed by their exclusion from the monophyletic cluster that assembles mosquito-borne and tick-borne flaviviruses in phylogenetic trees  from Culex sp. However, these sequences share only 76.1% of sequence identity (as defined by Blast2 sequence comparison), clearly below the 84% limit defined by Kuno and others [Anopheles mosquitoes, four of which could be classified to the species level as An. (Cel) pretoriensis and An. (Ano) coustani.One of the other lineages of cISF identified is represented by a viral sequence obtained from d others  and, theMansonia-specific cISF/NS5Δ in Fig. Ma. africana, Ma. uniformis and Cx. antennatus, were cloned and sequenced. Once again, the obtained sequences fell within the same monophyletic cluster. Moreover, when the structure of these DNA fragments was investigated, all of them revealed a similar architecture  the presence of an amplicon with approximately 0.5 kbp. This amplicon was observed in association with 31/351 (8.8%) of the pools analyzed, by itself in 9/351 (2.6%) or in combination with the expected 1 kbp DNA fragment in 22/351 (6.3%). However, given its size, it would correspond to a deleted form of the NS5 coding gene suggesting (i) that it might have been amplified from defective viral genomes and/or (ii) rearranged forms of retro-transcribed viral DNA, possibly integrated in mosquito genomes as previously observed –35, and/Alphavirus-specific primers [Very different results were obtained when either  primers  or those primers  were useBunyavirales, a decision was made not to restrict the screening of bunyaviruses to RVFV, but to extend it, in a smaller subset (43/351) of the pools of mosquitoes collected in different geographic areas of Mozambique, using Phlebovirus and Orthobunyavirus primers [Ae. (Adm) fowleri (n = 1), Ae. (Dic) adersi (n = 1), Ae. (Muc) sudanensis (n = 1), Ae. (Neo) circunluteolus (n = 4), Ae. (Neo) mcintoshi (n = 2), Ae. (Ste) aegypti (n = 1), Ae. (Ste) metallicus (n = 1), An.(Ano) coustani (n = 2), An. (Ano) tenebrosus (n = 1), An. (Cel) funestus (n = 1), An. (Ano) ziemani (n = 1), An. (Cel) pharoensis (n = 1), An. (Cel) pretoriensis (n = 2), Cq. (Coq) metallica (n = 1), Cx. (Cux) antennatus (n = 5), Cx. Cux) tritaeniorhynchus (n = 2), Cx. (Cux) neavei (n = 2), Cx. (Cux) pipiens s.l. (n = 2), Cx. (Cux) poicilipes (n = 1), Cx. (Cux) zombaensis (n = 1), Cx. sp. (1), Ma. (Mnd) africana (n = 3), and Ma. (Mnd) uniformis (n = 4), Mimomyia (Mim) mimomyiaformis (n = 1), and one pool of Ae. (Neo) sp.On the other hand, given the overwhelming diversity of the viruses that compose the recently proposed Order  primers , 39. ThiOrthobunyavirus genomes, in 5 pools, two of An. coustani , two of An. pretoriensis  and one of Cx. tritaeniorhynchus (sequences LC 461994 and LC46195) mosquitoes, a DNA fragment with the expected size was, indeed, amplified. All these amplicons were sequenced, but while BLASTn/x sequence searches did indicate a viral origin, unexpectedly they did not seem to have derived from bona fide Phlebovirus genomes, and this was confirmed by phylogenetic analysis using an assemblage of Phlebovirus, Bandavirus, Banyangvirus, and Goukovirus reference sequences. Regardless of the fact that the Phlebovirus group was paraphyletic, the sequences obtained from the analyzed mosquitoes from Mozambique did not cluster in any of the viral taxa in the tree, but rather formed 3 independent genetic lineages, as indicated by the arrows in Supplementary Fig. Bunyavirales. The obtained results  were put in contact with sub-confluent monolayers of C6/36 cells for virus isolation. Viral replication was allowed for 14 days (one blind passage was carried out at the end of the first week), after which the presence of viral genomes was verified by RT-PCR using the same primers used for viral screening. At day three after the first blind-passage, three of these supernatants revealed evident CPE that included an apparent cell growth arrest accompanied by cell rounding, detachment from the flask surface, and sometimes clumping  or suggested  to circulate in Mozambique , 42, 43.squitoes –14.Aedes mosquitoes which were clearly underrepresented in our screening. Furthermore, no Orthobunyavirus sequences were ever detected in a small sample of the pools analyzed .The molecular screening that was carried out did not reveal the presence of RVFV or any recognizable pathogenic alphaviruses, bunyaviruses or flaviviruses. These include viruses such as DENV, ZIKV, CHIKV, o’nyong nyong, Sindbis or Middelburg . While tOn the contrary, the use of a highly degenerate flavivirus-specific primer set  confirmeNS5 sequences were apparently associated with multiple species from 4 genera, supporting the perception that cISF are widespread in the natural populations of mosquitoes. Some of these NS5 sequences seemed to segregate away from previously described viral assemblages and formed isolated branches in phylogenetic trees. Others were joined in clusters with multiple operational taxonomic units that were never exclusively associated with a single species of mosquitoes, adding to the possibility that cISF may not host species-restricted [Culex origin for one of the sequences (LC462017), given the fact that it was almost identical to many others amplified from Mansonia mosquitoes, its association with Culex mosquitoes is disputable. Furthermore, although a molecular confirmation of the identity of these mosquitoes was obtained by COI-sequence analysis, the sequencing strategy used (Sanger) is a population-approach that only reveals the sequence of the most abundant molecular form in a PCR-product, while minor variants fail to be detected. Therefore, we cannot formally exclude the possibility that sequence LC462017 may have been derived from one/a small number or even body-parts of non-Culex mosquitoes (possibly Mansonia) originally present in the pool in question.The different genetic lineages of viral stricted , 48. HowMansonia spp. (n = 29) in one pool of Anopheles sp. and another of Culex sp. mosquitoes, the flavivirus-specific primers used generated a smaller than expected PCR product, with approximately half the size (≈0.5 kbp). The analysis of some of these smaller amplicons showed that they corresponded to defective versions of the RdRp coding sequence and their origin was found to be DNA (vDNA), rather than RNA. For all those cases where a nucleotide sequence could be obtained, a shared ancestry between the latter and bona fide viral NS5 sequences (obtained by RT-PCR) was also revealed.Surprisingly, in a high number of pools of Aedes mosquitoes [Anopheles could not be obtained due to technical difficulties, the fact that virtually identical vDNA sequences could be amplified from DNA extracts of Mansonia and Culex mosquitoes is hard to explain given the evolutionary divergence of these taxa. Moreover, while these vDNA forms could result from exposure of these mosquitoes to a common source of viruses, the possibility of a contamination of pools of Culex mosquitoes with even a limited amount of the highly abundant Mansonia specimens, cannot be discarded.While we cannot ascertain, at this stage, whether the flavivirus vDNA forms are present as part of the host genome (endogenized), or whether they exist in the form of a stable extra-chromosomal DNA element, flavivirus-like sequences have been known to occur in the genome of mosquitoes for over a decade, especially in association with squitoes , 34. WhiNS5 sequences found in this study seem to exclude the possibility that translation of an RNA transcribed from them might result in an active protein. In any case, they could participate in the establishment of persistence viral infections by controlling the siRNA response, as previously suggested [Whereas the presence of bacterial symbionts of mosquitoes can alter the competence of mosquitoes for transmission of pathogenic viruses , to whatuggested .Phlebovirus sequences, the observation of a specific amplicon in association with 5 pools of 2 different species of Anopheles (An. coustani and An. pretoriensis) and one species of Culex (Cx. tritaeniorhynchus) mosquitoes suggested that these viruses might have been detected. However, the different phylogenetic analyses were congruent in showing (i) their inclusion in the Phenuiviridae family, (ii) but their exclusion from the Phlebovirus genus, (iii) and their separation into three genetic lineages. Two of these sequences did segregate in a stable monophyletic cluster defining a genetic lineage with no assigned designation, but that included sequences previously detected in other studies [Given the a priori specificity of the primers used for the screening of  studies , 40, 41,Mansonia and Culex mosquitoes clearly dominate the collections in the 3 provinces of Mozambique that were the focus of this study. However, pathogenic flavivirus such as the Spondweni virus (the closest known relative to ZIKV), have indeed been isolated from Ma. africana and Ma. uniformis [Culex quinquefasciatus mosquitoes in Haiti [Mansonia sp. mosquitoes include the S. Louis encephalitis and West-Nile viruses , alphaviruses , orthobunyaviruses [Although no recognizable pathogenic viruses were identified in the course of this work, this may result from a combination of multiple factors that include sampling bias. In fact, collections did not focus on settings where DENV/ZIKV/CHIKV were previously known to circulate in Mozambique , 42, 43,niformis , as wellin Haiti . Associaaviruses , and phlaviruses .Mansonia spp [While sampling bias may partially explain the absence of some of the arboviruses that have been previously shown to circulate in Mozambique, other factors may also explain the results obtained. These include a low natural incidence of arboviruses in the areas where mosquitoes were collected, or the concurrent absence of recorded cases of human/animal disease cases associated to the circulation of viruses such as RVFV during the mosquito collection periods. Furthermore, a technical limitation of this study is associated with the use of a less technologically advanced virus detection approach based on convectional RT-PCR, as opposed to addressing viral screening with a bona fide metagenomic experimental design combined with the use of NGS sequencing methods. To the best of our knowledge, this study and the previous detection of ISF in onia spp , are theThe attempted isolation of some of the viruses identified in this work in insect cells was not successful. This fact may probably result from a combination of factors that include the use of only one blind passage and a single cell-line. Indeed, while C6/36 cells have been extensively used for the isolation of ISVs they may not be susceptible and well as permissive to all insect viruses. In this regard, it should be added that some ISF seem to be restricted to theirs hosts , and thitaxon-specific primers.The nature of these viruses is currently under investigation. Furthermore, bioinformatics investigations for producing the unidentified CPE observed in inoculated cells is encouraged for better understanding the prevalence of insect-specific viruses in many genera of mosquitoes , 55. WhiThis study reports for the first-time novel insect-specific flaviviruses and phenuiviruses, as well as frequent flavivirus-like viral DNA forms in several widely known vector species. While a large diversity of ISVs have been found on a global scale , 58 in a2-baited miniature CDC-light traps. These mosquitoes were stored in dry ice, and then transported to the laboratory for sorting and taxonomic identification using keys proposed by Gillies and Coetzee [A total of 14,519 mosquitoes were collected in rural settings in Mozambique (located in west southern Africa) between November 2014 and December 2015 as part of the work of Abílio, AP (In Preparation) at Massingir (in the province of Gaza), Namaacha (in the province of Maputo), and Mopeia (in the province of Zambézia)  buffer. After 3 pulses of 1 min (with 30 s breaks on ice), the mosquito macerates were clarified by centrifugation, as previously described . RNA, as well as DNA, were extracted from 200 μl of clarified mosquito homogenate using NZYol® , as indicated by the supplier. The extracted RNA was dissolved in 30 μl nuclease-free water, while the obtained DNA sediments were dissolved in 40–100 μl using a 1:1 mixture of 8 mM NaOH and TE buffer .The extracts of total RNA served as a template for the synthesis of cDNA, that was carried out with the NZY First-Strand cDNA Synthesis Kit  using random hexamers, and a thermal profile including 10 min at 25 °C, 45 min at 52 °C and 10 min at 80 °C (for enzyme inactivation), followed by treatment with RNaseH (20 min at 37 °C).NS5 sequences  was carried out using previously described primers and reaction conditions [nsP4 gene  was used to detect the presence of the genomes of alphaviruses [Detection of flavivirus nditions . A generaviruses , while Raviruses . Finallyaviruses  and the aviruses , or as daviruses . Nelorpiaviruses . All theAedes (Ste) albopictus C6/36 cell line was used for virus isolation. Cells were maintained at 28 °C (in the absence of CO2) in L-15 Leibovitz Medium  supplemented with 10% heat-inactivated fetal bovine serum (FBS) , 2 mM L-glutamine , 100 U/ml penicillin and 100 μg/ml streptomycin  and 1 × tryptose phosphate broth . Approximately 500 μl of filter-sterilized mosquito homogenate were diluted in the same volume of phosphate buffered saline (PBS), and inoculated onto semi-confluent layers of C6/36 cells grown in T25 culture flasks . After 1 h at room temperature , the viral inoculum was removed, 5 ml of L-15 Leibovitz Medium (2% FBS) was added to each flask, and the cell cultures were incubated at 28 °C for a week. Culture supernatants collected after a single blind-passage were used as viral stocks and stored at − 80 °C. Cytopathic effect (CPE) was determined by microscopic observation of the inoculated cell cultures.Multiple alignments of nucleotide (nt) or amino acid (aa) sequences were performed using the iterative G-INS-I and E-INS-I methods as implemented in MAFFT vs. 7  followedhttp://beast.bio.ed.ac.uk/tracer) was used to diagnose stationarity and adequate (> 300) effective sample size (ESS). The trees were logged on every 5000th MCMC step, and the tree sample was summarized using TreeAnnotator v1.8.3 as maximum clade credibility (MCC) trees, with median heights used as the node heights in the tree, after discarding 10% of them as burn-in. The FigTree v1.4.2 software was used to visualize the phylogenetic trees (http://tree.bio.ed.ac.uk/software/figtree/).Phylogenetic trees were constructed using both Maximum Likelihood (ML) and Bayesian approaches. The best-fitting evolutionary models used were those suggested by JModeltest2  and W-IQ-tree  for the analysis of nt  or aa alignments . Phylogenetic analyses based on the ML optimization criterion were carried out using the Mega 6.0 software , and thehttp://www.boldsystems.org/).The molecular confirmation of the morphological identifications of mosquitoes was carried out on the basis of the analysis of the barcoding section  essentially using Bold Systems-v4 (available at https://blast.ncbi.nlm.nih.gov/Blast.cgi).The nt sequences obtained in the course of this study were deposited in the GenBank/ENA/DDBJ databases under accession numbers LC461994-LC462019, and LC-462246-LC462257, and LC517270-LC517293. The reference sequences used for analyses presented in this manuscript where directly downloaded from the public sequence databases. Whenever necessary, nt sequence similarity searches were carried out using BLASTn, and BLASTx , or after infection (day 3) with viruses present in three independent pools of Ma. uniformis, An. ziemani, and An. pretoriensis mosquitoes collected in Mozambique.Additional file 2.Additional file 3: Supplementary Table 1. PCR primers and thermal profiles used in this work.


Vickermania, a new genus of fly midgut-dwelling parasites that bear two flagella in contrast to other trypanosomatids, which are unambiguously uniflagellate.The family Trypanosomatidae encompasses parasitic flagellates, some of which cause serious vector-transmitted diseases of humans and domestic animals. However, insect-restricted parasites represent the ancestral and most diverse group within the family. They display a range of unusual features and their study can provide insights into the biology of human pathogens. Here we describe Vickermania has an odd cell cycle, in which shortly after the division the uniflagellate cell starts growing a new flagellum attached to the old one and preserves their contact until the late cytokinesis. The flagella connect to each other throughout their whole length and carry a peculiar seizing structure with a paddle-like apex and two lateral extensions at their tip. In contrast to typical trypanosomatids, which attach to the insect host’s intestinal wall, Vickermania is separated from it by a continuous peritrophic membrane and resides freely in the fly midgut lumen.Vickermania developed a survival strategy that relies on constant movement preventing discharge from the host gut due to intestinal peristalsis. Since these parasites cannot attach to the midgut wall, they were forced to shorten the period of impaired motility when two separate flagella in dividing cells interfere with each other. The connection between the flagella ensures their coordinate movement until the separation of the daughter cells. We propose that Trypanosoma brucei, a severe human pathogen, during its development in the tsetse fly midgut faces the same conditions and follows the same strategy as Vickermania by employing an analogous adaptation, the flagellar connector.We propose that  Flagellates of the family Trypanosomatidae (Euglenozoa: Kinetoplastea) are obligatory parasites inhabiting vertebrates, arthropods, leeches, and plants , 2. SincFor a long time, the classification of monoxenous trypanosomatids was based solely on morphology, with each genus characterized by a predominant and/or unique morphotype , 13. BecHerpetomonas muscarum is the first named monoxenous trypanosomatid, originally reported in 1856 from the housefly Musca domestica in the USA  of speed) being a proxy for agility Fig. . Expectezoobank.org:pub:73A2FCA2-F35B-4050-8EE7-DCAFCAEBD770.Since this publication contains nomenclatural changes, it has been registered in Zoobank: urn:lsid:Class: Kinetoplastea Honigberg, 1963Order: Trypanosomatida Kent, 1880Family: Trypanosomatidae Doflein, 1901Vickermania Kostygov et Yurchenko, 2020 gen. nov.Genus: zoobank.org:act:F31B2BA7-A896-49B8-A5CF-4A6F3BD4FB6FZoobank LSID: urn:lsid:Diagnosis: promastigotes with two anteriorly oriented flagella of unequal length, typically attached to each other, separated only during cell division; uniflagellate cells appear only shortly after division, as the second flagellum starts growing soon after it; flagellar tips have rounded or elongated apex and lateral extensions; large and loosely arranged kDNA.Herpetomonas muscarum ingenoplastis Rogers and Wallace, 1971 now classified as Vickermania ingenoplastisName-bearing type: Etymology: The generic name honors Prof. Keith Vickerman, an outstanding British protistologist, who focused on studies of various parasitic protists, mainly trypanosomatids.Vickermania ingenoplastis  comb. nov., stat. nov.Species diagnosis and description: up to 80% cells in culture are biflagellate; cells are 27.3–40.7 μm long and 2.3–3.5 μm wide; flagellum is 28.8–44.5 μm long; kDNA mass is about 2.1 μm long, 0.6 μm in diameter and has grape seed shape; it has loose periphery, and dense kernel.Phormia regina Type host: Localization: midgutType locality: Minneapolis–Saint Paul, Minnesota, USA.Type material: the culture ATCC30259.Gene sequences: MT241904 (18S rRNA gene), and MT248932 (gGAPDH gene), KX901631 (draft genome).Herpetomonas sp., which was lost as a result of long-term cultivation; (ii) the culture F72 isolated from Lucilia sericata  in Oksochi, Novgorod Oblast, Russia , belongs to the same species according to the 18S rRNA gene sequences.Comments: (i) opisthomastigotes and short promastigotes reported in the original description  were notVickermania spadyakhi Ganyukova et Kostygov sp. nov.zoobank.org:act:D68B3910-9D91-402B-8FEC-2CF9AF1506AF.Zoobank ID: urn:lsid:Species diagnosis and description: up to 60% cells in culture are biflagellate; cells are 22.5–75.9 μm long and 1.8–4.7 μm wide; flagellum is 25.6–64.0 μm long; barrel-shaped kDNA mass with diameter of 0.6–0.8 μm at both ends and 1 μm in center.Nemopoda nitidula (Diptera: Sepsidae)Type host: Localization: midgutType locality: Sob’ railway station, Yamalo-Nenets Autonomous Okrug, Russia Type material: the culture S13, deposited in the research collection of Parasitic Protists of the Zoological institute RAS .Etymology: the specific epithet has Russian origin and means “from near Padyakha”, referring to the local name of the river Sob’, close to which the species was collected.Gene sequences: MT241903 (18S rRNA gene), and MT248930 (gGAPDH gene).Comment: the type culture was established from an insect with a mixed infection, including morphologically dissimilar epimastigote-type cells with a distinct phylogenetic position.Since the description of biflagellate trypanosomatids more than a century ago by Stanislav Provázek, the discoverer of epidemic typhus , their vVickermania is not truly biflagellate in the sense that it lacks a tetraflagellate stage prior to the cell division, but instead becomes transiently uniflagellate right after its completion. However, these trypanosomatids bear two flagella throughout most of their cell cycle and, in this respect, shall be considered biflagellate. In order to achieve this, Vickermania had to disconnect the well-established ancestral coordination of the cell cycle, and this results in a relatively high percentage of anomalous forms associated with various cell division disturbances.Our study confirmed that Vickermania become biflagellate? Taking into account that the main function of the flagellum is locomotion [Vickermania preserves a connection between the two flagella for as long as possible. This allows them acting as a single entity, not interfering with each other due to possible uncoordinated beating. Our analysis of motility confirmed that the cells with a single or two joint flagella are significantly faster and more agile as compared to those with two separate flagella. The observed low frequency of cells with separate flagella in the log-phase and its increase only in degrading post-stationary cultures further supports this assumption. In order to build the new flagellum in time, the trypanosomatid starts growing it in the very beginning of the cell cycle. The peculiar “seizing” structure of the flagellar tips likely mediates a firm attachment and a proper orientation of the growing flagellum.Why did then comotion , this iscomotion , VickermVickermania than to other trypanosomatids? As highly diverse and successful parasites, trypanosomatids evolved a range of strategies allowing them to colonize a wide range of hosts [Vickermania thrives in the midgut, it cannot implement attachment, since in its fly host the midgut wall is separated from the lumen by a semipermeable continuous structure known as the peritrophic membrane [Leishmania spp. can penetrate this barrier during their development in sandflies [Vickermania to rely only on an active movement against the flow created by intestinal peristalsis. In such harsh conditions, dividing cells are vulnerable due to their decreased motility. Indeed, in the strain CP021, which showed lower infection efficiency in the model fly species , the proportion of dividing cells displayed a dramatic drop during development in the gut. Since bearing two separate flagella in such conditions can make cells vulnerable, Vickermania has shortened this period. Regrettably, very little is known about the development of other monoxenous trypanosomatids inhabiting the fly midgut [Why may motility be of higher importance to of hosts . The evoof hosts , which aof hosts –58. Althmembrane . It is pmembrane . Attachmandflies . Howeverandflies . A coexiy midgut , and theTrypanosoma brucei live in the endoperitrophic space of the tsetse fly midgut, where they also have no chance to attach to the intestinal wall. Interestingly, this developmental stage demonstrates a surprising analogy to Vickermania, as its newly growing flagellum is also connected by the tip to the old one [T. brucei is different: it developed for this purpose a specialized structure called the flagella connector, which is absent from all other developmental stages [T. brucei eventually migrates from the intestine into the salivary glands, where it becomes attached to the epithelium, while Vickermania is doomed to permanent movement.The procyclic trypomastigotes of  old one . Howeverl stages . This stl stages , but in Vickermania represents overlooked, yet widely distributed trypanosomatids that adapted to the life in the fly midgut, where the default trypanosomatid strategy, namely the attachment to the intestinal epithelium, is impossible. Instead, they rely on constant movement, preventing discharge from the host gut due to intestinal peristalsis. Therefore, these parasites shortened the period of the cell cycle, when they bear two separate flagella interfering with each other and thus become vulnerable. This is achieved by disconnecting the flagella duplication from cell division and by developing a mechanism to join the new growing flagellum with the old one. Our findings also shed light on the biology of the deadly human pathogen Trypanosoma brucei. During development in the tsetse fly midgut, it likely faces the same conditions and follows the same strategy as Vickermania. The flagella connector, unique for the gut stage of T. brucei, and the peculiar seizing structure at the tip of the Vickermania flagellum developed independently, but play the same role ensuring efficient motility in dividing cells. These remarkably analogous adaptations demonstrate the plasticity of one of the main eukaryotic cellular structures, the flagellum.The new genus H. m. ingenoplastis was obtained from the Fiocruz Protist Culture Collection. It is a replicate of the type culture ATCC30259, isolated from Phormia regina  [Nemopoda nitidula (Sepsidae) collected in July 2016 near Sob’ railway station , Yamalo-Nenets Autonomous Okrug, Russia, as described elsewhere [Lucilia sericata  captured in May 2019 in the village Oksochi , Novgorod Oblast, Russia. To date it could not be purified from the accompanying fungal contaminants.The CP021 culture of horidae) . The cullsewhere . The intAll trypanosomatid cultures were maintained at 23 °C in Schneider’s Drosophila medium  supplemented with 10% fetal bovine serum (FBS) , 500 μg/ml of streptomycin and 500 Units/ml of penicillin (Sigma-Aldrich), and passaged weekly. In order to study growth dynamics, the cultures CP021 and S13 were seeded in hexaplicates with a starting concentration of 40,000 cells/ml and subsequent counting with hemocytometer at days 1, 2, 3, 5, 7, 9, and 11.Calliphora vicina and Lucilia sericata , maintained at the Zoological Institute, St. Petersburg, since 2010. The insects were cultivated at 25 °C, with larvae being fed pork liver, while adults received sugar cubes and clean water. Two days after hatching from pupae, 50% of individuals were used for experiments, while the remaining half underwent control dissection and were always trypanosomatid-negative. Before the experiments, the flies were kept without water for 24 h.Experimental infections were performed using laboratory cultures of the flies The thirsty flies were given access to a bowl containing flagellates in cultural medium, which was replaced after 24 h with clean water. From that moment, the time of infection was counted and the dissections were performed on days 1, 3, 5, and 15. The possibility of intraspecific horizontal transmission was tested by placing 5 uninfected flies with 5 recently (1–2 days) infected ones. Ten days later, all insects were dissected.In order to assess the duration of single infections, individual infected flies were isolated in plastic tubes of a diameter that prevented them from turning around and, thus, being re-infected with their own feces. The tube opening was closed with a fine-meshed net, through which the fly had access to a piece of cotton wool soaked in 17% sugar syrup. At the posterior end, the tube contained a piece of dry cotton wool absorbing excreta . For the visualization of axonemes, primary mouse monoclonal anti-α-tubulin and secondary anti-mouse IgG-TRITC goat antibodies  were used. After washing with PBS and staining with either DAPI  or SYTO24 Green Fluorescent Nucleic Acid stain (Thermo Fisher Scientific), slides were observed under Leica DM2500 fluorescent microscope. The samples from cultures and midguts of infected flies were prepared and processed for scanning and transmission electron microscopy as described previously [The trypanosomatid cells on Giemsa-stained smears were photographed using DM 2500 Leica microscope and measured with Fiji software . For flueviously , 68, 69.Total genomic DNA from cultures and one field sample was isolated with the DNeasy Blood & Tissue Kit . 18S rRNA and gGAPDH genes were amplified using the primer pairs S762/S763  and M200The phylogenetic position of the studied trypanosomatids was inferred based on the analyses of 18S rRNA and gGAPDH genes commonly used for this groups as well as more powerful phylogenomic analysis.Seventy-four 18S rRNA gene sequences of various trypanosomatids including the two species under study  with two separate flagella and (ii) with a single or two joint flagella. Videos were recorded at a 720 × 480 pixels resolution with frame rate of 30 fps and then cut into 100 (50 per cell category) 3-s clips for each cell to be analyzed. The cells were selected according to the following criteria: (i) moving actively; (ii) showing no signs of cytotomy, abnormal size, or cell body deformation; and (iii) appearing in the examination area for at least 3 s. The frame-wise coordinates of each cell central point were obtained with the Manual Tracking plugin in Fiji software and used to calculate the following parameters: (i) average speed; (ii) maximum displacement, i.e., maximum distance between any two points on the trajectory; (iii) speed standard deviation (SD); and (iv) relative speed SD, i.e., SD of speed changes expressed as a percentage of an average value. Since all obtained per-category distributions significantly differed from normal as judged by Additional file 1: Figure S1. Cells on a Giemsa-stained smear from the xenic culture F72. a Cell appearing as monoflagellate. b Unambiguously biflagellate cell.Additional file 2: Table S1. Measurements (in μm) of trypanosomatid cells in three cultures (N = 50). A-N and A-K are the distances between the anterior end of the cell and nucleus or kinetoplast, respectively.Additional file 3: Figure S2. Growth dynamics of the cultures CP021 and S13 with values averaged from six independent biological replicates. Confidence bars indicate standard deviations.Additional file 4: Figure S3. Maximum likelihood phylogenetic tree based on gGAPDH gene sequences. Numbers at nodes indicate posterior probability and bootstrap percentage, respectively. Values below 0.5 or 50% are replaced with dashes or omitted. The tree is rooted with the sequence of Paratrypanosoma confusum. All well-supported clades of described subfamilies or genera are collapsed. The scale bar denotes number of substitutions per site. The two species of biflagellate trypanosomatids studied here are highlighted.Additional file 5: Figure S4. Flagellates of the strain S13 in the gut (SEM). Abbreviations: tr, trypanosomatids; pm, peritrophic membrane. Scale bar: 20 μm.Additional file 6: Table S2. Summary statistics for motility parameters in two cell categories and results of statistical tests.Additional file 7: Table S3. Correlation of motility parameters in two cell categories.Additional file 8: Figure S5: Three-second trajectories of the cells of the category 1.Additional file 9: Figure S6: Three-second trajectories of the cells of the category 2.Additional file 10: Figure S7. Individually isolated flies in infection longevity experiments.Additional file 11: Table S4. Sequences of 18S rRNA gene used in this work.Additional file 12: Table S5. Sequences of gGAPDH gene used in this work.Additional file 13: Table S6. Genomic sequences used in this work.


Increased concentration of thyroid antibodies, i.e., TPOAb, occurred more frequently in patients with TSH ≥ 2.5 mIU/L than those with TSH < 2.5 mIU/L . The same differences were found in the group of 240 patients after excluding those on L-thyroxine treatment. Blood lymphocyte concentration was the only independent linear parameter associated with TSH ≥ 2.5 mIU/L  but only in the group of 280 patients with normal thyroid tests. TSH concentration correlated positively with blood lymphocyte  and TPOAb  concentrations but only in the group of 280 patients with normal thyroid tests. Less favorable lipid profiles and a higher prevalence of thyroid antibodies in women of reproductive age with high-normal TSH suggests that L-thyroxine treatment should be considered in such patients. The significance of a positive association between high-normal TSH and blood lymphocytes requires further evaluation.High-normal TSH can be associated with metabolic abnormalities and infertility. Aims of this study are to analyze retrospectively if routinely measured blood laboratory and anthropometric parameters differ between women of reproductive age with TSH < 2.5 mIU/L and with TSH ≥ 2.5 mIU/L. Retrospective analysis was performed in 466 female inpatients, aged 13–51, hospitalized in an endocrine department. The group of 280 patients with normal thyroid tests  was selected and it was divided into two subgroups, i.e., with TSH < 2.5 mIU/L and TSH ≥ 2.5 mIU/L . After excluding patients on L-thyroxine treatment (n = 240), those with TSH ≥ 2.5 mIU/L constituted 22.92% (n = 55). In the group of 280 patients with normal thyroid tests, an abnormally high concentration of triglycerides and an abnormally low HDLC/cholesterol ratio occurred more frequently in women with TSH ≥ 2.5 mIU/L than those with TSH < 2.5 mIU/L (17% vs. 7%,  Thyroid tests depend on age. TSH increases with age and is thus found in lower normal ranges in younger subjects ,2. It isIt has been documented in several studies that high-normal TSH is associated with some adverse effects concerning reproduction and with certain abnormalities in metabolic processes. Concerning the former, either preconception  or firsTaking into account the above observations, new evidence is required to document that high-normal TSH in women of childbearing age may be associated with some pathological processes and, therefore, should be treated as abnormal.The aim of this study was to retrospectively analyze if routinely measured blood laboratory parameters as well as anthropometric parameters differ between women of reproductive age with TSH < 2.5 mIU/L and those with TSH ≥ 2.5 mIU/L.The procedures used in the study were approved by the Ethical Committee of the Polish Mother’s Memorial Hospital—Research Institute, Poland [No. 40/2018].Retrospective analysis was performed in 466 female inpatients, aged 13–51, hospitalized in 2016 and 2017 with a suspicion of different endocrine diseases , thyroid dysfunction included, in the Department of Endocrinology and Metabolic Diseases, Medical University of Lodz, Poland. Only patients, in whom endocrine entities (apart from thyroid dysfunction) were not confirmed were enrolled into this retrospective analysis.The following laboratory parameters were measured: total cholesterol, HDL cholesterol (HDLC), LDL cholesterol (LDLC), HDLC/cholesterol ratio, triglycerides (TGs), glucose, insulin, insulin resistance index (IRI), C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), vitamin D, iron, and complete blood count, i.e., red blood cells (RBC), hemoglobin (Hgb), white blood cells (WBC), neutrophils, lymphocytes, and platelets. Anthropometric measurements included body height, body mass, and BMI.t-test , and free triiodothyronine (FT3), and thyroid antibodies, i.e., thyroid peroxidase antibodies (TPOAbs), thyroglobulin antibodies (TgAbs), and TSH receptor antibodies (TSHRAbs), were evaluated in morning blood samples from all inpatients. Two hundred and eighty patients with normal thyroid tests  were selected. They were divided into two subgroups, i.e., 214 subjects with TSH < 2.5 mIU/L (Controls), and 66 subjects with TSH ≥ 2.5 mIU/L, the latter constituting 23.6% of the whole sample examined. No statistically significant differences were found between subgroups concerning age and body mass index (BMI) when evaluated by Student’s unpaired t-test .Thyroid antibodies were abnormal in 42 patients: only TPOAbs were abnormal (≥ 34 IU/mL) in 15 patients; only TgAbs were abnormal (≥ 115 IU/mL) in 10 patients; both TPOAbs and TgAbs were abnormal in 16 patients, and all three thyroid antibodies were abnormal (TSHRAb ≥ 1.75 IU/L) in one patient. Forty patients were on L-thyroxine replacement therapy (25–150 µg daily) due to previously diagnosed hypothyroidism; two of them were diagnosed after subtotal thyroidectomy due to either non-toxic or toxic nodular goiter, and both were in the group with TSH < 2.5 mIU/L.All patients enrolled into retrospective analysis neither suffered from any entity affecting thyroid tests, nor obtained medication which could have interfered with laboratory methods to measure thyroid tests. None of them obtained medication that can affect the lipid profile.Exclusion criteria constituted exposure to ionizing radiation or to any other potential prooxidative agent, alcohol consumption, cigarette smoking, and any diagnosed acute or chronic disease , apart from diagnosed and properly treated thyroid dysfunction.Thyroid tests  and thyroid antibodies  were measured in blood serum with an immunochemiluminescent method . Other laboratory parameters were measured in the blood with standard methods .t-test. The results are presented as means ± SEM. Univariate and multivariate logistic regression analyses were used to determine which continuous variable might have been associated with TSH ≥ 2.5 mIU/L. For the evaluation of the correlation among particular parameters, Pearson’s correlation coefficient was used. The two-sided ratio comparison test was used to evaluate the frequency of events. Statistical significance was determined at the level of p < 0.05.The data were statistically analyzed using Student’s unpaired 3/μL) in all patients. In the group of 240 inpatients (after excluding 40 patients on L-thyroxine replacement), no statistical differences were found between the subgroups with TSH < 2.5 mIU/L and those with TSH ≥ 2.5 mIU/L concerning all linear parameters (data not shown).Patients with TSH ≥ 2.5 mIU/L constituted 23.6% (n = 66) of all patients with normal thyroid tests (n = 280) or 22.92% (n = 55) of patients after excluding L-thyroxine treatment (n = 240). Mean values of all linear parameters in a subgroup with TSH < 2.5 mIU/L and in the subgroup with TSH ≥ 2.5 mIU/L, evaluated in the group of 280 inpatients with normal thyroid tests are presented in The percentage of abnormal lipid profiles differ between subgroups concerning TGs concentrations and HDL/cholesterol ratio. Either in the group of 280 inpatients with normal thyroid tests b or in tConcerning thyroid antibodies, increased concentrations of TPOAbs and TgAbs occurred more frequently in subjects with TSH ≥ 2.5 mIU/L, when the whole group (n = 466) of patients was considered a. Howevep < 0.001). However, in the group of 240 patients , this association of blood lymphocytes and TSH ≥ 2.5 mIU/L lost its statistical significance.Among all measured linear parameters, blood lymphocyte concentration constituted the only linear variable statistically associated with TSH ≥ 2.5 mIU/L, when evaluated by the univariate regression analysis. Thus, the second step of regression analysis, i.e., the multivariate regression analysis, was not performed, and blood lymphocyte concentration was proven to be the only independent factor associated with TSH ≥ 2.5 mIU/L . Similarp = 0.031) and with TPOAb concentrations , and TSH concentration correlated negatively with FT4 concentration , when the group of patients with normal thyroid tests was considered (n = 280), but not after excluding patients on L-thyroxine treatment.TSH concentration correlated positively with blood lymphocyte concentration , between lymphocyte concentration and TgAb concentration , as well as between lymphocyte concentration and TSHRAb concentration  in patients with normal thyroid tests (n = 280).At the same time, however, no correlations were found between lymphocyte concentration and TPOAb concentration  strongly suggests that most of our patients with high-normal TSH are just unhealthy, therefore they require replacement therapy with L-thyroxine. Thus, it seems that there is a huge-scale phenomenon concerning the prevalence of subclinical hypothyroidism in women of childbearing age.The key finding of our study is the association between high-normal TSH and the abnormal lipid profile in women of childbearing age. As was mentioned in the Introduction, such an association has already been partially documented in the literature. In this context, it is worth mentioning that an abnormal lipid profile during gestation affects unfavorably the course of pregnancy and progeny. For example, high TGs and low HDL cholesterol at late gestation are independent predictors of macrosomia in women without diabetes mellitus . AdditioConcerning thyroid antibodies, they occurred more frequently in subjects with TSH ≥ 2.5 mIU/L. Thus, a higher prevalence of thyroid antibodies in patients with high-normal TSH suggests that this group of patients is at very high risk of developing overt hypothyroidism. Again, this observation supports the need for replacement therapy in women with TSH ≥ 2.5 mIU/L. In this context it is worth mentioning that TPOAb positivity has recently been documented to be predictive of a reduced live birth rate in patients with recurrent pregnancy loss; expectedly, L-thyroxine replacement improved live birth rate . TPOAb pA new finding in our study is the association between TSH levels and peripheral blood lymphocyte concentration. Although the absolute number of lymphocytes remained in normal ranges in all subjects, the mean lymphocyte concentration was higher in patients with TSH ≥ 2.5 mIU/L, lymphocyte concentration correlated positively with TSH, and lymphocyte concentration was the only independent factor associated with high-normal TSH. At the same time, lymphocyte concentration was not associated with the level of antibodies. These results suggest that the absolute number of lymphocytes depends directly on thyroid function but it does not depend on autoimmune processes. This positive association between TSH and lymphocyte levels, both remaining in normal ranges, cannot be clearly explained at this moment. However, it can be speculated that this relationship results from an abnormal lipid profile in patients with high-normal TSH. Such a hypothesis can be supported by recently published observations performed concerning patients with type-2 diabetes mellitus in whom proatherogenic parameters of lipid profile correlated positively with the lymphocyte level .It is worth mentioning that different results have been published recently showing an association between high-normal TSH and abnormal values of certain parameters, the significance of which is not clear. For example, it has been observed that serum TSH is positively associated with microalbuminuria in euthyroid diabetic patients and the mechanism of this interesting relationship is currently the subject of speculation .It is well known that reference ranges for thyroid tests depend also on the method used. However, whereas reference intervals for the main thyroid hormone, i.e., thyroxine, are confirmed to be method-related, the reference ranges for TSH do not differ significantly between methods . Taking In conclusion, in women of reproductive age with normal thyroid tests, TSH ≥ 2.5 mIU/L is associated with a less favorable lipid profile and with a higher prevalence of thyroid antibodies. These results support our standpoint that high-normal TSH at reproductive ages can be considered abnormal in most patients and, therefore, the replacement therapy with L-thyroxine should be taken into account in such cases, especially with a coexisting abnormal lipid profile or positive thyroid antibodies. Further studies should be performed to find additional benefits from adjusting TSH to the lower end of normal ranges in women of reproductive age. The significance of the positive association between so-called normal TSH levels and normal blood lymphocytes is unknown at this moment and requires further evaluation.


Thrice-weekly haemodialysis is the usual dose when starting renal replacement therapy; however, this schedule is no longer appropriate since it does not consider residual renal function. Several reports have suggested the potential benefit of beginning haemodialysis less frequently and incrementally increasing the dose as the residual renal function decreases. However, all the data published so far are from observational studies. Thus, this clinical trial avoids any potential selection bias and will assess the possible benefits that have been observed in observational studies.2 are eligible. Patient recruitment began on 1 January 2017 and will last for 2 years or until the required sample size has been recruited to ensure the established statistical power has been reached. The minimum follow-up period will be 1 year. Anuric patients with acute renal failure and patients who return to haemodialysis after a kidney transplant failure are excluded. It has been calculated that 44 patients should be recruited into each group to achieve a power of 80% in a two-sided comparison of means with a usual significance level of 0.05. A time-to-event analysis will estimate the probability of kidney function survival in both groups using the Kaplan–Meier method. Survival curves will be compared with log-rank tests. This survival analysis will be complemented with a proportional hazard model to estimate the hazard ratio of kidney function survival adjusted for any confounding factors. Analyses will be carried out in accordance with the intention-to-treat principle.This report describes the study protocol of a randomized prospective multi-centre open-label clinical trial to evaluate whether starting renal replacement therapy with twice-weekly haemodialysis sessions preserves residual renal function better than the standard thrice-weekly regimen. We also explore other clinical parameters, such as concentrations of uremic toxins, dialysis doses, control of anaemia, removal of medium-weight uremic toxins, nutritional status, quality of life, hospital admissions and mortality. Only incident haemodialysis patients who can maintain a urea clearance rate KrU ≥ 2.5 mL/min/1.73 mThe incremental initiation of dialysis may preserve residual renal function better than the conventional treatment, with similar or higher survival rates, as reported by observational studies. To our knowledge, this is the first clinical trial to evaluate whether initiating renal replacement therapy with twice-weekly haemodialysis sessions preserves residual renal function better than beginning with the standard thrice-weekly regimen.NCT03302546. Registered on 5 October 2017.ClinicalTrials.gov,  However, adherence to this scheme is low, based on data in the Dialysis Outcomes Practice Patterns Study [The usual schedule is thrice-weekly haemodialysis sessions, and only a few patients receive dialysis less frequently . Data frns Study  and on cns Study .Periodically measuring the renal urea clearance rate is not common practice in haemodialysis units because renal function is considered to decrease rapidly after the onset of dialysis treatment. This could, in part, justify the poor implementation of lower-frequency haemodialysis. However, in recent years there has been growing interest in less frequent dialysis schemes , 4–8, thBeginning haemodialysis less frequently and incrementally increasing the dose as residual renal function decreases is referred to as incremental or progressive haemodialysis –7, 11. M2 to indicate when to increase the frequency of haemodialysis. This program has had satisfactory results and no clinical side effects in our previous experience [The Haemodialysis Unit of the Nephrology Department of Hospital Universitario Ramón y Cajal is a pioneer in implementing an incremental haemodialysis program, having more than 10 years of experience , 7, 17. perience , 7, 17.The percentage of patients who start renal replacement treatment with two weekly sessions has increased from 28% in 2006 to 84% in 2017. Currently, about 25% of the patients in our unit are treated with this twice-weekly frequency , 7, a loHowever, note that published studies on incremental haemodialysis are observational, with potential selection bias , 15, 18.To our knowledge, this is the first clinical trial to evaluate whether initiating renal replacement therapy with twice-weekly haemodialysis sessions preserves residual renal function better than beginning with the standard thrice-weekly regimen. Recently, the study protocol has been published of another clinical trial that assesses whether incrementally starting with one session of haemodialysis per week reduces mortality in incident patients compared with patients who start renal replacement therapy with the conventional method .Our hypothesis is that initiating renal replacement therapy with twice-weekly haemodialysis sessions preserves residual renal function better than the conventional regimen of beginning with thrice-weekly sessions. In addition, we hypothesise that maintaining renal function provides beneficial clinical effects, such as better control of anaemia, greater removal of medium-weight uremic toxins, and better nutritional status and quality of life, and has a safety profile like that of the conventional schedule.The main outcome is loss of kidney function, defined as urine volume output less than 100 mL in 24 h, 12 months after the initiation of haemodialysis therapy.The secondary outcomes are: (1) erythropoietin dosage, (2) beta-2 microglobulin concentration, (3) hydration and nutritional status, (4) concentrations of uremic toxins, (5) dialysis dose, (6) number and duration of hospital admissions, (7) mortality and (8) quality of life.In addition, cost-efficiency will be assessed by recording the cost of haemodialysis sessions and the monthly consumption of erythropoietin.2. As treatment masking is not feasible, treatment allocation is centralized for all participating hospitals, and performed by an agent external to the researchers. Once a participating centre has verified that a patient meets the inclusion criteria, the randomization centre will provide the treatment group allocation. The investigators will not know the randomization sequence. The two groups will be those beginning with two haemodialysis sessions per week (2HD) and those beginning with three haemodialysis sessions per week (3HD).This is a randomized prospective multi-centre open-label clinical trial. Randomization will be stratified in every recruitment centre, ensuring a 1:1 ratio between the two study groups in all centres. The trial compares the initiation of renal replacement therapy with two haemodialysis sessions per week to the conventional three sessions per week in patients with residual urea clearance rate KrU ≥ 2.5 mL/min/1.73 mThe participants are incident patients at five university hospitals in the Community of Madrid (Spain): H. U. Ramón y Cajal (coordinating centre), H. U. del Henares, H. U. La Paz, H. U. Príncipe de Asturias and H. U. Clínico San Carlos. The minimum follow-up period will be 1 year.2. Before enrolment, participants must sign the informed consent form.Prospective participants must have stage 5 chronic kidney disease, must have started chronic haemodialysis and must be able to maintain a residual urea clearance rate KrU ≥ 2.5 mL/min/1.73 mAnuric patients, patients with acute renal failure, and patients who return to haemodialysis after a kidney transplant failure will be excluded from the trial., recover renal function or transfer to another facility or if they withdraw their informed consent.Patients will be withdrawn from the study if they receive a renal transplant, dieThe following will be assessed at the momento fo enrollment: age, sex, renal disease, Charlson comorbidity index, urine volume output in 24 h (mL), urea clearance (mL/min), creatinine clearance (mL/min).Kt / V), erythropoietin dosage (units per week per kg of weight), hydration, nutrition, hospital admissions, days of admission, causes of hospitalization and number of haemodialysis sessions.The following will be assessed at the start of haemodialysis treatment (baseline) and every 2 months during a follow-up of at least 1 year: urine volume output in 24 h (mL), urea clearance (mL/min), creatinine clearance (mL/min), haemoglobin (g/dL), haematocrit (%), creatinine (mg/dL), urea (mg/dL), Na (mmol/L), K (mmol/L), albumin (g/dL), pre-albumin (mg/dL), calcium (mg/dL), phosphorus (mg/dL), parathyroid hormone (pg/mL), ferritin (ng/mL), beta-2 microglobulin (mg/L), brain-derived natriuretic peptide (pg/mL), C-reactive protein (mg/L), dose of dialysis  / V Total andKt / V = dialysis Kt / V + (5.5 KrU) / V ,Total V is 58% of the dry weight (Watson formula) [where formula)  and KrU 2), lean tissue index (kg/m2) and fat tissue index (kg/m2).Hydration and nutrition are measured by multi-frequency electrical bioimpedance . The following metrics are collected: overhydration (L), amount of extracellular water (L), amount of intracellular water (L), extracellular / intracellular ratio, body mass index , at 6 months and at 12 months (close-out). This is a validated questionnaire for the haemodialysis population.The schedule of enrolment, intervention and assessments is shown in Fig. Kt / V. The 2HD group will start with two sessions of 4 h.The dialysis technique used for each patient  will be maintained throughout the follow-up period. The 3HD group will start with three sessions lasting 3–3.5 h to achieve an adequate dialysis Kt / V should be 1.6 or 1.2 for 2 sessions per week and 3 sessions per week, respectively. Modifications to the dialysis procedure, such as increasing the blood flow, bath flow, dialyzer surface or duration of the dialysis session, will be made if patients do not reach an adequate Kt / V.According to Casino and López , to obta2 in two determinations made in a 2-week interval, or they develop uncontrolled hypertension, heart failure, or a clinical event that indicates that the number of haemodialysis sessions should be increased.Participants in the 2HD group will be switched to the 3HD group if their urea clearance rate falls below 2.5 mL/min/1.73 mPatient recruitment began on 1 January 2017 and will be extended until the required sample size has been recruited to ensure the established statistical power has been reached. The minimum follow-up will be 1 year.Considering the data published in observational studies , 5, 7 anThe data collected will be stored in a database specially designed for this study using Access (Microsoft Office Access 2007). The data will be entered by a dedicated member of the project team, who will ensure the integrity and quality of the data. The statistical analyses will be carried out with the statistics program Stata v.14 .p < 0.0051. The final analysis with the whole trial population will be carried out at the end of the follow-up at a significance level of 0.04795.We will perform a blind interim analysis when at least half of the patients have been recruited and followed for at least 1 year. We will stop the trial if the group difference in the main outcome is statistically significant at a significance boundary t-test) or nonparametric tests (Mann–Whitney U test). The associations of categorical variables with the two treatment groups will be assessed by chi-squared tests or Fisher exact tests, as appropriate.For the univariate analysis of quantitative variables, we will check the normality of the data using the Kolmogorov–Smirnov test. Depending on the assumption of normality, the two arms will be compared using parametric tests  is a quantitative and objective parameter. Other possible limitations may be due to insufficient recruitment such that the analysis population differs from the population treated in standard clinical practice.The transition from pre-dialysis to renal replacement therapy is a crucial moment for patients with chronic renal failure and has significant clinical, social and occupational impacts . AlthougAn incremental approach to the initiation of dialysis may allow some patients to retain more of their residual renal function with an improved quality of life and similar or higher survival rates than those receiving the conventional treatment, as reported by observational studies , 19, 28.However, all previous studies have been observational, and prospective trials, such as this one, are required to determine, without selection bias, the potential benefits and safety of the incremental schedule –11, 28, 2, without excluding a priori patients with severe comorbidities. In our experience, more than 80% of patients who started incremental renal replacement treatment did not suffer from any relevant complications [This randomized clinical trial compares twice- versus thrice-weekly haemodialysis sessions for patients who can maintain a residual urea clearance rate KrU ≥ 2.5 mL/min/1.73 mications . For the


Liriodendron tulifipera L. belongs to Magnoliaceae which is one of the basal angiosperm families. To understand intra-species variations on chloroplast genome in Liriodendron genus, we presented complete chloroplast genome of L. tulifipera, which is 156,387 bp long and has four subregions: 85,606 bp of large single copy (LSC) and 18,778 bp of small single copy (SSC) regions are separated by 26,002 bp of inverted repeat (IR) regions including 129 genes . The overall GC content of the chloroplast genome is 37.0% and those in the LSC, SSC, and IR regions are 34.9%, 30.5%, and 42.8%, respectively. Twelve single nucleotide polymorphisms (SNPs) located in one region and one insertion and deletion are found between two L. tulifipera genomes. INDEL Phylogenetic trees show that two L. tulifipera chloroplasts are clustered together and are sister to Magnolia species. Liriodendron is one of the genera belonging to Magnoliaceae, which is basal angiosperm family . Genome sequencing was performed using HiSeq2000 at Macrogen Inc., Korea. Chloroplast genome was assembled and confirmed by Velvet 1.2.10 (Zerbino and Birney L. tulifipera chloroplast genome (NC_008326).Total DNA of L. tulifipera (Genbank accession is MK477550) is 159,885 bp long (GC ratio is 39.2%) and has four subregions: 85,606 bp of large single copy  and 18,778 bp of small single copy  regions are separated by 26,002 bp of inverted repeat , which is longer than that of L. tulifipera by 1 bp  and one insertion and deletion (INDEL) are identified. This amount of intra-species sequence variations is larger than those of Coffea  and maximum likelihood (bootstrap repeat is 1000) trees using MAFFT 7.388 (Katoh and Standley L. tulifipera chloroplasts are clustered together and are sister to Magnolia species (Fourteen Magnoliaceae containing two  species .


Plasma exchange (PE) and immunoadsorption (IA) are standard therapeutic options of immune-mediated neurological disorders. This study evaluates the relation of the relative quantity of applied dose of PE and/ or IA and its achieved therapeutic effectiveness within the treated underlying neurological disorders. In a retrospective study, we evaluated data from PE and IA carried out 09/2009-06/2014 in neurological patients at the University-Hospital of Saarland, Germany. Apheresis dose was defined as the ratio of the extracorporeal treated plasma volume to the patient’s plasma volume. Effectiveness was assessed through disease-specific tests and scores by the attending neurologist(s); results were classified into response or no response. 1101 apheresis , in 153 hospital-stays were carried out, averaged, 7.0 treatments per patients, 82% responded, 18% not. Mean applied apheresis dose per treatment was 0.91 with mean doses of 1.16 for PE and 0.81 for IA. The totally applied mean dose per stay was 5.6 . No correlation was seen between apheresis dosing and treatment effectiveness . PE and IA in therapy-refractory immune-mediated neurological disorders majorly achieved a measurable severity improvement – without correlation to the applied dose. Moreover, our data rather suggest, that effectiveness may be given with volumes below currently recommended volumes. Central pathogenic elements of these immune-mediated diseases are B-lymphocytes and antibodies as directed to structures of the nervous system and are involved in the process of neuroinflammation, leading to neurological symptoms e.g the ascending paralysis or sensory deficits. So far, recommendations for relative plasma volume per patient and treatment are few and inconsistent. In our study, we exactly ask the question of a direct proportional effectiveness of PE and IA regarding response/ no response correlated to the applied apheresis dose, meaning the relative quantity of treated plasma volume.Apheresis is an established method of removing proteins and antibodies from the blood, here of interest plasmapheresis (PE) and immunoadsorption (IA), two methods with different targeted blood components. In PE, the patient’s plasma is withdrawn and replaced by fresh frozen plasma or by protein-contending solutions. In IA, the patient’s plasma is passed through an adsorber, eliminating the target components. PE and/ or IA are, at least in therapy-refractory casesAll patients with study relevant immune-mediated nlogeuroical disorders and treated in the Department of Neurology at the Saarland University Medical Centre (UKS) between Sept 2009 and June 2014, were per se eligible for this study. Thereof, the therapeutic option of apheresis was considered in case of therapy failure of steroids, immunoglobulins or in critically ill patients; the final indication for apheresis was provided by the Department of Neurology. Whereas, the choice of apheresis procedure (PE or IA) was determined interdisciplinary, based on the neurological disorder, individual risk factors and comorbidities of each patient. As this study evaluates clinical data purely retrospectively, written informed consent from all patients included was not obligatory, according to the vote of the local ethics committee . All clinical study data were obtained through revision of medical documentation and entered anonymously into our data bank.All study incorporated apheresis treatments, PE and IA, were carried out by the Department of Nephrology UKS. For this purpose, the multifunctional device Octo Nova® was used. For IA, a Tryptophan-Immunadsorber (Immusorba® TR-350) from Diamed was used. In 4 cases the immunoadsorption device ADAsorb  and the proteinA-adsorber Immunosorba® were used.The actual study population was divided into three groups: patients treated by PE, treated by IA, or both – PE and IA – while reasons for change of procedure(s) were e.g., pre-existing treatment with ACE inhibitors, absolute contraindication for IA or allergic reactions on components of the donor plasma during PE.Apheresis dose was defined as the actual volume of plasma either exchanged during PE or treated during IA related to individual patients’ body plasma volumes. Actually exchanged or treated plasma volumes were taken from apheresis protocols; patient plasma volumes (PPV) were calculated according to Sprenger as following: Plasma volume [l] = 0.065 × body weight [kg] × (1-hematocrit). The single treatment relative apheresis dose is defined as the quotient of PE/ IA volume over patient plasma volume, while the cumulative administered apheresis dose per patient and hospital stay is the sum of all then applied treatment doses divided by the patient’s plasma volume. Both, effectiveness and clinical benefit were evaluated through disease-specific tests and scores by the attending neurologist. Effectiveness, classified into yes/ no response, was assessed for each hospitalisation. Both the administered single dose ratio per treatment as well as the sum dose ratio per hospital stay were correlated to the overall clinical benefit = yes/ no response.Currently two options for anticoagulation are available: firstly, systemic anticoagulation with heparin , or regional anticoagulation with citrate, infused to blood volume with a ratio of 1:32 and target concentration of post-filter ionized calcium 0.25–0.35 mmol/l as described by Calatzis [13].Complications were classified into severe, moderate and mild complications; accordingly severe resulted partly in a complete abortion while other severe up to moderate led to discontinuation of single or pause of overall apheresis treatment with/ without potential drug therapy; mild complications often allowed to continue apheresis treatment, led seldomly to spontaneous stop, some were treated by a short-term mediation therapy.2 and Overall Disability Sum Score (ODSS)3 before and after PE and/ or IA treatment. In responders, at least one of these scores was ameliorated afterwards. There against, no responders showed no improvement or even worsening of ODSS and MRC sum scale.The effectiveness of PE and/ or IA was evaluated through determination of CIDP and GBS, MRC Sum Score4 was assessed, and responders’ EDSS score was at least 0.5 points lower after apheresis treatment. In myasthenia gravis, the besinger score5 was determined, and responders showed score improvement. In rare conditions (e.g. stiff person syndrome) without established scores yet available, the treating neurologists assessed potential benefits of apheresis to the patients.In more detail and/ or examples: In optic neuritis, visual acuity was examined by an ophthalmologist, here responders showed improvement of visual acuity after treatment. In cases of severe multiple sclerosis relapses, the expanded disability status scale (EDSS)Continuous variables are expressed as mean ± SD  or as median and the range minimum to maximum (skewed variables). Categorical variables are presented as a percentage, unless otherwise stated. For comparison(s) between continuous variables the unpaired t-test was used. Categorical variables were compared by use of Chi-square test or Fisher exact test, respectively. Two-sided p-values <0.05 were considered as statistically significant. Data analysis was performed using IBM SPSS Statistics 19™ .During study period overall 3425 patients with immune-mediated neurological disorders within this study’s focus were treated: CIDP (n = 478), GBS n = 41), myasthenia gravis (n = 305) and multiple sclerosis (n = 2601), in 102 patients  PE or IA was carried out. One patient case was excluded due to insufficient available data. Overall, in 101 patients then 1101 procedures during 153 hospital stays (in-patients) were carried out; some patients were admitted to hospital more than once. Of 153 in-patients 31 were treated with PE, 102 with IA, and 20 with PE and IA. Basic characteristics of the final in-patient study population are presented in Table , myasthe: plasma exchange and immunoadsorption and choice of anticoagulation) For anticoagulation mainly heparin was used. Mean applied dose of heparin was 5.965IE (1.000 – 17.000IE) with higher amounts in PE compared to IA (6835 vs. 5680IE). Overall, regional anticoagulation was used in 36/153 in-patients, in detail in 155/1101 therapeutic apheresis. In 16 in-patients both, heparin and citrate were applied. In one case of suspected but not confirmed HIT II argatroban was used for anticoagulation in 3 IA.Distribution of anticoagulation is shown in Fig. 182% responded to apheresis during hospital compared to 18% non-responders as shown in Fig. Mean apheresis dose per treatment was 0.91 (0.21 to 1.72) times the patients’ plasma volumes. During one stay cumulative apheresis dose was 5.6 (0.47 to 64.8) times the PPV as shown in Fig. Mean apheresis dose of each PE  was higher than of each IA . In contrast, cumulative apheresis dose was lower for PE  compared to IA .2 = 0.014). There was also no correlation between cumulative treatment dose per stay and effectiveness (coefficient of correlation R2 = 0.003). Mean apheresis doses per stay in patients with or without response were 5.9 and 5.4 times the patients’ plasma volume. Moreover, the effectiveness of applied apheresis doses was independent from chosen apheresis procedures (PE or IA or both).In a nutshell, apheresis dose per treatment was not correlated to effectiveness. Mean apheresis doses per treatment in patients with clinical response or no clinical response were 0.89 and 0.97 times the patients’ plasma volume  270 complications occurred, in some cases 2 complications occurred simultaneously. Severe complications were rare in 3 of 270 (1.1%), moderate complications occurred in 42 of 270 (15.6%) and most complications were mild with 225 of 270 (83.3%). Interestingly, complications occurred two times more often in PE 37% (88/238 PE) compared to IA 18.1% (156/863 IA).: complications in PE or IA–4 A: complications occurring for IA, 4B: complications occurring for PE).In a similar way, complications were more often under anticoagulation with citrate compared to under heparin (38.9% versus 19.5%). Occurrence of different complications is shown in Fig. 4A,B6. Today, TPE is an established method for the treatment of immune-mediated neurological disorders1. However, there are only few and inconsistent recommendations with respect to the relative quantity of plasma volume to be treated. This study shows that the applied apheresis dose ratio does not seem to influence like-wise proportionally the actual effectiveness of therapeutic apheresis in immune-mediated neurological disorders.Therapeutic plasma exchange (TPE) was described for the first time in 1914 as an extracorporeal blood purification technique7. In the early 1980s, new techniques of plasma separation then started to allow treatments with higher total volumes as such8.Historical development of TPE supports this finding as therapeutic apheresis has already been applied in the sixties with total volumes of 500-700 ml, independently from patients’ plasma volumes9. But, plasma exchange against human albumin containing solutions is restricted in its relative maximum due to the loss of proteins, complement and coagulation factors limiting the TPE frequency and lowering the achievable/ possible reduction of antibodies9. However, in the 1980s higher exchange volumes were described with 1.5-2 fold plasma volume8. In contrast, currently therapeutic apheresis is recommended 5-7 times within 10-14 days, with replacement volumes between 1 and 1.5 times the patients’ plasma volumes10. However, as far as our literature research revelled current relative volume recommendations have not yet been evaluated in prospective studies of considerable size on apheresis dose and its proportional effectiveness, even less on immune-mediated neurological disorders as of interest here. The few studies with a mentionable number of patients (68 to 419) and treatments (388 to 1945)14 rather report about quite heterogeneous underlying diseases and varying doses. These studies do not allow to draw clear conclusions for relative dose recommendations regarding effectiveness per se and/ or proportionality.In principle, studies show that antibody removal is subject to a saturation curve with a relative optimum at a 1-fold plasma volume exchange, meaning removing about 50% of the antibodiesNow, our current study is not only based on one of the largest numbers of evaluated TPEs and patients but additionally purely assesses a restricted category of underlying diseases, solely immune-mediated neurological disorders.13. Moreover, there was no correlation between the applied relative volume per treatment and/or the relative cumulative dose during one hospital stay and the proportional effectiveness of apheresis as such – instead, the study cohort could be divided into responders and non-responders per se.Following the ASFA recommendations, an average of 7.1 treatments was carried out within our study cohort, while the mean apheresis dose (0.91 times PPV) was lower than suggested. Though, the positive apheresis response rate of 82% within these otherwise standard therapy-refractory cases was comparable to other studies15, showed comparable effectiveness to the usually applied doses (1-1.2 times PPV)16. Another study evaluated the impact of the number (not volume!) of treatments in GBS on effectiveness, showing firstly an improvement and then a saturation curve: Increasing treatments from 2 to 4 in mild and moderate cases resulted in higher effectiveness, but an increase in the severe cases up to more than 4 treatments did not achieve any additional benefit17. A third case showed as well effectiveness of apheresis in myasthenic crisis despite treatment volumes below recommendation18.A few small studies indicate similarly, that effectiveness of apheresis may be also given with volumes below recommendation: PE with treated plasma volume of 0.6 times the patients’ plasma volume in optic neuromyelitis19. Higher plasma treatment volumes can be reached e.g. with proteinA-adsorber allowing treatments of high volumes (1.5 to 2.5 times PPV). However, removal of proteins limits the frequency and total volume of application of IA: a therapy volume of 2.5 litres, reduces antibodies by 65–75%, fibrinogen by 75% and albumin by 25%20. The reduction of fibrinogen leads to pronounced hypofibrinaemia in 20% of IA treatments21. An increasing number of studies report about successful application of IA in standard therapy-refractory cases of immune-mediated neurological disorders. However, effectiveness of PE and IA was evaluated only in a few studies so far showing comparable effectiveness18 in different neurological disorders with clinical benefit in 71% in steroid-refractory cases22 to up to 100% in patients with neuromyelitis optica23. Although our evaluation is retrospective, positive response rate to IA is comparable with studies reporting about PE.Overall, in literature treatment of immune-mediated neurological disease is predominantly described with PE. But, removing pathological antibodies from the patient’s blood is also possible with IA. Here, due to saturation of the binding sites, the plasma volume being effectively treated with most plasma-adsorber, is limited to volumes of 2 to 2.5 litresAlthough our study is limited due to the retrospective design a correlation between apheresis dose ratio and clinical benefit should have been visible with respect to the high number of patients and treatments. The treated plasma volume is varying due to technical reasons, complications or clinical evolution.Therapeutic apheresis is a promising therapeutic tool in therapy-refractory cases of immune-mediated neurological disorders. PE and IA show comparable clinical benefit in our study, independently from the relatively applied apheresis dose. Our results, such as others, seem to show increasing evidence that the needed apheresis dose for effectiveness in treatment responders is below the historically grown dose expectations and actual recommendations. Therefore, our results are promising and should be confirmed in prospective studies.