TRIM8 gene mutations have been reported as the genetic basis of autosomal dominant (AD) neuro-renal syndrome in children, which presents with epileptic encephalopathy, focal segmental glomerulosclerosis (FSGS), developmental delay, and mental retardation. In this study, we report the cases of two children with significant proteinuria due to de novo nonsense mutations of the TRIM8 gene.TRIM8 gene .Case 1 was a 7-year-old girl who presented with proteinuria and developmental delay, and her renal biopsy showed FSGS. She developed end-stage renal disease (ESRD) 3 years after onset. Case 2 was another 7-year-old girl who developed proteinuria only at age 3, and renal biopsy showed glomerular segmental mesangial proliferative lesions. The two girls underwent genetic testing but we did not find a positive result in the whole exon. However, cluster analysis revealed two new nonsense mutations of the We reported the clinical manifestation of this neuro-renal syndrome for the first time in China. It is necessary to perform genetic testing in children with steroid-resistant significant proteinuria to identify its etiology and avoid the side effects of immunosuppressants. TRIM8 exon 6. Recent studies have suggested that the mutation of the TRIM8 gene is the genetic basis for the early onset of neuro-renal syndrome  and nephrotic range proteinuria (24-h proteinuria: 1.007–1.820 g). The blood biochemistry was normal except for a slightly low albumin level of 36.1 g/L and a mildly high total cholesterol level of 5.87 mmol/L. Other laboratory results, such as levels of anti-nuclear antibodies, anti-double stranded DNA antibodies, hepatitis B virus, and human immunodeficiency virus were negative, and renal ultrasound showed no obvious abnormalities. At 3 years and 10 months of age, her percutaneous renal biopsy demonstrated segmental glomerular sclerosis (1/10) categorized as global obsolete (2/10), and immunofluorescence staining showed IgM trace, which suggested focal segmentary glomerulosclerosis (FSGS) . Her treA 7-year-old Chinese girl was born at full term. Her father was healthy, while her mother was diagnosed with FSGS and renal stones, and her grandfather also had renal stones. After she was born, there were no signs of motor or language retardation. She had proteinuria and microscopic hematuria during a routine examination at the age of 3 years and 4 months. Her physical tests were negative including heart, lung, liver, and nervous system examinations. The urine dipstick test was 1+-3+ positive for protein with 24–84 red blood cells/HPF, and 24-h protein excretion was 1.7 g. The levels of serum creatinine, albumin, anti-double stranded DNA antibodies, anti-nuclear antibodies, and complements were all within normal limits. At 3 years and 5 months of age, renal ultrasound showed a hypoechoic cortex and no renal stones. Renal biopsy revealed glomerular segmental mesangial proliferative lesions, and immunofluorescence staining was IgA+ . After tTRIM8. The genome coverage and the average depth of coverage were 99% and 134.89 × in case 1 and 99% and 132.66 × in case 2, respectively. However, the cluster analysis in 2021 found two de novo nonsense mutations in exon 6 of the TRIM8 gene. Case 1 showed c.1461C>A, p.Tyr487* and case 2 showed c.1453 C>T, p.Gln485*  in 2019 but we did not find an obvious pathogenic mutation. WES covers the coding sequence of nearly 20,000 functional genes containing all exons of .Gln485* . Accordide novo LOF (loss of function) mutation of TRIM8, and then we found that TRIM8 is highly expressed in the renal according to the Genotype-Tissue Expression (GTEx) database, which suggests that it may be related to renal disease. Therefore, we searched our database and found that these two girls with mutations in exon 6 of the TRIM8 gene had similar phenotypes.Cluster analysis is a mathematical-statistical method to classify research subjects according to the information provided by the research subject, which is used widely in genetic testing datasets . It is ade novo frameshift mutation in the TRIM8 gene . , 9. TRIMTRIM8 mutations, and the specific mechanism needs further study.The two mutations we reported in this study were located in amino acids 485/487, including the truncated mutations in exon 6 that were at the C-terminal of the TRIM8 protein. According to structural analysis, TRIM8 conducts homodimerization through its coiled helical- structure, and its C-terminal is necessary for correct nuclear localization. The mutation leading to translation termination is located after the NLS, causing the sequence length to shorten. This shortened sequence may be detrimental to the stability of nuclear localization. Weng et al. found that the mutated TRIM8 protein was distributed diffusely in the nucleoplasm and could not be limited to the nuclear bodies . TypicalCompared with previously reported cases, only case 1 of these two patients had significant motor retardation, normal language development and intelligence, and no seizures. However, both cases showed a renal phenotype presenting with nephrotic-range proteinuria. Renal biopsy in case 1 showed FSGS, progression to ESRD within 3 years, and initiation of peritoneal dialysis. This conformed to the characteristics of the previously reported cases. Case 2 had mild symptoms but did not respond to steroids, and a variety of immunosuppressive therapies could not achieve a complete remission or maintain remission. She maintained 0.3–0.6 g/24 h proteinuria with normal renal function, indicating that patients without a neural phenotype may have a better prognosis, but further research is needed to verify this hypothesis. According to the previous studies, most patients (13/19) progressed to ESRD, and 6 of them received renal transplantation. None of the five patients with 24-year follow-up data had clinical recurrence . This shTRIM8 gene mutation is a rare childhood-onset AD disease representing significant proteinuria and FSGS accompanied by epilepsy and developmental delay. Most patients have a poor prognosis, so early diagnosis and treatment are necessary. Genetic testing and cluster analysis are helpful in finding patients with atypical phenotypes.In conclusion, a neuro-renal syndrome caused by The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by Clinical Trials Ethics Committee of the Chinese People's Liberation Army General Hospital of Eastern Theater Command. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin. Written informed consent was obtained from the minor(s)' legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.XL, YW, MW, LJ, ZS, XY, TJ, QK, ZX, and CG drafted the manuscript or revised it critically for important intellectual content, provided final approval of the version to be published, and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work were appropriately investigated and resolved. All authors contributed to the article and approved the submitted version.Funding was provided by the Pediatric Medical Innovation Team of Jiangsu Province (CXTDA2017022).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


ZIP protein  is an important metal transporter that transports Zn, Fe, and other divalent metal ions in plants. In this study, we identified 20 ZIP genes in lettuce (Lactuca sativa L.). We used bioinformatics methods and renamed them according to their E value in hmmsearch. We also analyzed their gene structure, chromosomal location, constructed a phylogenetic tree, conserved motifs, performed synonymous analysis and responses to abiotic stresses. The results show that these LsZIP genes have 3–11 exons and were distributed unequally on 8 of the 9 chromosomes in lettuce. Based on phylogenetic analyses, the LsZIP gene family can be divided into three subfamilies, and the LsZIP genes within the same subfamily shared similar gene structure. The LsZIP genes contain 12 Motifs, of which Motif1 to Motif8 are widely distributed in group Ⅰ. Furthermore, the LsZIP gene contains numerous hormones and anti-stress response elements. Real-time quantitative PCR demonstrated that most LsZIP genes is up-regulated under the elemental stress in this experiment, indicating that they are positively regulated. But different elemental stressors can induce the expression of LsZIP gene to varying degrees. The LsZIP genes also change in response to different elemental stresses. The present study serves as a basic foundation for future functional studies on the lettuce ZIP family.The  ZIP  is an important metal transport protein, found in many organisms, including bacteria, fungi, animals and plants. It contains a variety of metal substrate-specific membrane transport proteins and has been shown to be involved in the transfer and absorption of divalent metal ions by plants [ZIP proteins have similar membrane topology and eight potential transmembrane domains. They are rich in conserved histidine residues and variable region contains potential metal binding domains. There is a variable region between TM-3 and TM-4, and the outer surface of the plasma membrane contains amino- and carboxyl-terminals [ZIP genes were first identified in Arabidopsis thaliana in plants.The gene family y plants . In geneerminals . ZIP genZIP genes have been shown to be expressed in the absence of zinc (Zn) to low levels of Zn ions. Among them are AtZIP1 and AtZIP3, which are expressed in roots. AtZIP4 transports Zn ions into cells or between plant tissues [ZIP transporters such as AtZIP1, AtZIP2, AtZIP3 and AtZIP4 are functionally transporters with different metal affinities [ZIP transporters are directly involved in the accumulation of Zn in edible plant parts [ZIP family genes in plants shows a dynamic pattern. For example, under conditions of Zn deficiency, some ZIP transporters are more highly expressed, but when the Zn levels return to normal or high, their expression is lower. Therefore, when Zn is added to a medium with Zn deficiency levels, their expression decreases within 2 hours [ZIP genes are involved in the uptake and transport of Zn in different plant parts. In rice, overexpression of OsZIP1 increases the accumulation of Zn and iron in roots, shoots, and seeds [OsZIP4 and OsZIP5 can significantly increase the Zn concentration in the roots of transgenic plants, but in seeds, the Zn content cannot be increased [TdZIP1 in wild wheat can lead to excessive accumulation of Zn in cells, resulting in Zn toxicity [ZIP is mainly responsible for the uptake and transport of Zn and Fe. However, these proteins may also play a role in the transport of other metals, such as Mn and Cu [ZIP gene is involved in the absorption and transportation of Cd. For example, in Chinese cabbage, ZIP2 and ZIP3 were found to be associated with the transport of Cd and showed high expression levels [SlZIP4 was positively correlated with Cd concentration in a certain concentration range in tomato [ZIP genes are also found in mulberry, rice, cole and tobacco, which are involved in the uptake and transport of Cd [Several finities . Studiesnt parts , 7. The  2 hours . Severalnd seeds , 10. On ncreased , 12. Simtoxicity . In genen and Cu , 15. In n and Cu , 17. Resn levels . The expn tomato . In addirt of Cd , 20–23.Lactuca sativa) is an important vegetable crop originally from the Mediterranean coast and cultivated worldwide [http://www.fao.org/faostat/en/#data/QC/visualize). Among the cultivated crop species, lettuce is known for higher Cd uptake, and higher accumulation in the leaves. Lettuce has a high rate of Cd uptake and transport. When grown on soil contaminated with Cd, it can quickly transport Cd to the buds [ZIP gene has been studied. However, there is no relevant research report on the ZIP protein in lettuce. Because of the importance of ZIP genes in metal ion uptake and transport and the specificity of lettuce in Cd uptake, it is reasonable to conduct a systematic study of the ZIP family in lettuce. With the completion of the genome sequencing of lettuce, we study the evolution of the lettuce ZIP gene family at the entire genome level and the expression tissue characteristics [Lettuce  from the Pfam protein family database (http://pfam.xfam.org/). The ZIP protein search was performed using the standard HMMER 3.0 software parameters from the Salad Gene database [ZIP genes that did not contain domains and duplicates [ZIP gene models. Then, the ProtParam tool (http://web.expasy.org/protparam/) was used to determine the sequence length, molecular weight, isoelectric point, and instability index of these putative LsZIP genes [LsZIP was determined using Wolf-PSORT (https://wolfpsort.hgc.jp/) [We downloaded the complete genome sequence databases of lettuce from the NCBI database (Genome v7: ID:6720851) and downloaded the hidden Markov model (HMM) file of the database –33. For plicates , 35. FinIP genes . Finallyhgc.jp/) .LsZIP genes, a multiple sequence alignment of 20 LsZIP sequences was performed using Clustal X software [LsZIP gene structures were examined using the Gene Structure Display Server (GSDS: http://gsds.gao-lab.org) to map the exon and intron composition. MEME online program (http://meme-suite.org/tools/meme) was used to identify conserved motifs of the lettuce ZIP protein with specific domains [To clarify the evolutionary relationship of 20 software . The LsZ domains . The optZIP protein sequences of 14 species  were downloaded from phytozome (www.phytozome.net). ClustalX was used to perform a multiple sequence alignment of these ZIP proteins (default parameters). Their phylogenetic tree was analyzed and constructed using MEGA7.0 software. The phylogenetic tree construction method used the Neighbor-Joining (NJ) method, and 1000 bootstrap replicates were filmed [LsZIP genes was also constructed using the same method and parameters. Besides, we predicted the secondary structure of lettuce ZIP protein using SOPMA software [ZIP protein based on the protein homology model in the swiss model database [According to the description in the literature, the e filmed . In contsoftware , and we database .LsZIPs under elemental stress, the PlantCARE Server (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) was used to extract the sequence 2000 bp upstream of the LsZIPs promoter to determind cis-acting elements. The results are visualized using GSDS.To identify the cis-regulatory changes in LsZIPs on 8 lettuce chromosomes were mapped and the Multiple Collinearity Scan toolkit (MCScanX), was used to analyze gene repetitions (default parameters) [LsZIP genes in evolution, TBTools software was used to calculate the non-synonymous (Ka) and synonymous (Ks) replacement rate of the LsZIP gene, and the Ka/Ks ratio was determined [Using the gene positions obtained from the lettuce gene database, 20 ameters) . Subsequtermined .-2*s-1, and irrigated with water. After a growth period of 7 days, they were transferred into a 1 litre hydroponic container containing 1/2 strength of Hoagland nutrient solution (pH = 6). Four, lettuce plants in each hydroponic container were grown for another week until the third leaf appeared, then the seedlings were transferred into a new container for treatment. Treatments were applied at two levels: of Zn-deficiency 0 mg/l and Zn-excess 10 mg/l; two levels: Fe-deficiency 0 mg/l and Fe-excess 200 mg/l; and one level of Cd 10 mg/l. The leaves were harvested after 0 h, 12 h, 24 h and 48 h. Three separate plants per point were used for each stress level, and these samples were quickly frozen in liquid nitrogen and then stored at -80°C.According to the results of previous experiments, the Cd-sensitive variety ‘lusu’ lettuce is the cultivar in this experiment . To ensuhttps://agbio.com.cn) was used for Total RNA. 400 ng total RNA and random primers were added to Evo M-MLV RT Premix for cDNA synthesis . qRT-PCR was performed using SYBR Green Pro Taq HS qPCR Kit . The online primer design  was used as a reference gene [SpeadyPure Plant RNA Extraction kit  were manually deleted. Finally, based on the presence of a complete ZIP domain on the surface, 20 gene models were selected and labeled as lettuce ZIP genes. The total 20 ZIP genes are distributed on chromosomes 1–8 and were renamed LsZIP1 to LsZIP20 according to their match values in HMMER search results. In this work, we identified 20 lettuce ZIP genes and divided them into three categories. The physical and chemical properties and subcellular location of the 20 LsZIP proteins are listed in LsZIP proteins, LsZIP19 was determined to be the smallest protein with 199 amino acids (aa), and the largest was LsZIP20 (578 aa). 18 LsZIP proteins are classified stable (instability index <40), while LsZIP12 and LsZIP14 are unstable (instability index> 40). The molecular weight of LsZIP protein ranges from 21.48 to 60.51 kDa, and the pI value ranges from 5.31 to 9.4. The predicted subcellular localization results show that 18 LsZIP proteins are localized in the Plasma membrane region and the remaining two proteins are located in the vacuole.Using the HMM file of the ZIP genes encoding protein sequences from 15 species and constructed a phylogenetic tree using the Neighbor Joining (NJ) method to determine their autologous or collateral relationships. As shown in Fig 2, the phylogenetic tree is divided into four clades. The genes of 8 monocot and 7 dicot plants are distributed among four clades. This indicates that the separation of the progenitor genes of the four clades occurred earlier than the separation of monocots and dicots. Among them, clade I and III can be further divided into two subclades, and clade IV can be further divided into four subclades. Except for IVa (contains only dicotyledonous plants) and IVc (contains only monocotyledonous plants), the remaining subclades contain both dicotyledonous plants and monocotyledonous plants. Twelve AtZIP genes are distributed among four clades, of which clade I contains two (not included in IB). Clade II contains one, clade III contains three, and clade four contains six (not included in IVc).We collected 133 ZIP genes in lettuce are unevenly distributed among the clade. Clade I has five lettuce ZIP genes, clade II has only one gene LsZIP13, clade III has five lettuce ZIP genes, and clade IV has nine lettuce ZIP genes. ZIP genes in lettuce are unevenly distributed across the clade. In all clades, the LsZIP gene is homologously related to the AtZIP gene. With the exception of LsZIP15 and 16 in Ia, LsZIP14, LsZIP18, and LsZIP20 in Ib, which show strong homology with monocots, the remaining lettuce ZIP genes resemble dicots. The homology is stronger. In clade IVb, five lettuce ZIP genes formed a unique monophyletic group, and LsZIP5 alone formed a unique monophyletic group. In the smallest clade group, lettuce ZIP genes are genetically related to Arabidopsis  and tomato , cucumber , and soybean (LsZIP13). More recently, this suggest that they may share a common ancestor. In addition, some LsZIP genes are arranged in pairs of smaller evolutionary branches, namely , as well as LsZIP8, LsZIP15 and LsZIP16 are distributed separately in evolutionary branches. This indicates that the lettuce ZIP gene family can spread by tandem duplication, segmental duplication, and transposition events. In the smallest clade, lettuce ZIP genes are genetically related to Arabidopsis  and tomato , cucumber  and soybean (LsZIP13). This suggest that they may have a common ancestor. To assess similarity information between lettuce ZIP proteins and ZIP proteins of other species and to comprehend the sequence conservation of ZIP proteins over evolution. ClustalX was utilized to analyze the multiple sequence alignment of 133 ZIP proteins from 15 species, the results are shown in ZIP proteins contain a total of seven transmembrane (TM) structural domains. In addition, there is a variable region between TM-3 and TM-4 containing a potential metal-binding domain enriched with histidine residues.ZIP family in lettuce, we mapped the exons-introns of 20 identified LsZIP genes. As shown in LsZIP genes are divided into three subfamilies, of which the I family is divided into three small subfamilies. In LsZIP genes were found to have 1 to 4 exons. LsZIP20 has 6 exons, LsZIP14 and LsZIP18 have 11 exons, while LsZIP19 has only one exon. In generally, the genes in the same group have similar structures. For example, all members of group Ib contain three exons. Through MEME motif analysis, 12 conserved motifs were found in LSZIP. The conserved sequences of the 12 motifs are shown in LsZIP15 and LsZIP16, motif 7, motif 9, and motif 12 were found in LsZIP15, and no motifs were found in Lszip16, LsZIP18 and LsZIP20. In general, LsZIP members of the same group have similar motif composition; for example, motif 1 to motif 8 are widely distributed in group I, while motif 10 and motif 11 are specific to group II. The clustered LsZIP pairs LsZIP5/8, LsZIP9/12, and LsZIP4/6 are even in their motifs. The similar motif arrangement among the LsZIP proteins in the subgroup suggests that the protein structure is conserved in a specific subfamily. Generally, LsZIP members in the same group have similar conserved motif composition and gene structure, which, together with the results of phylogenetic analysis, indicates that the group classification is reliable.To gain a deeper understanding of the evolution of the LsZIP proteins, we predicted their secondary and tertiary structures. The results of the secondary structure demonstrated that the percentages of α-helix, β-turn, random coil and extended strandwere 37.19% (LsZIP19) to 59.92% (LsZIP18), 1.75% (LsZIP4) to 7.44% (LsZIP20), 10.95% (LsZIP9) to 48.24% (LsZIP19), and 9.91% (LsZIP14) to 36.76% (LsZIP14), respectively , there was no significant difference in the 3D structures of the LsZIP family (containing 8 Alpha helix). Two proteins were identified in all LsZIP gene families, including the Bacillus bronchisepticus ZIP protein (BbZIP) and the membrane protein  and 17 salicylic acid responses (TCAGAAGAGG and CCATCTTTTT) occurred in 14 and 12 LsZIP gene promoter sequences, respectively. Four auxin-responsive cis-regulatory elements (GGTCCAT、AACGAC and TGACGTAA) are distributed in only four LsZIP genes, but 14 gibberellin-responsive cis-acting elements (CCTTTTG、TATCCCA and TATCCCA) are distributed in eight LsZIP genes. This is somewhat surprising because Zn is an essential element in the synthesis of tryptophan, and tryptophan is the precursor auxin synthesis. 36 MeJA-responsive cis-regulatory elements (TGACG and CGTCA) are distributed among 12 LsZIP genes, and 10 defense and stress-responsive cis-regulatory elements are distributed among 9 LsZIP genes.To further determine the role of the c stress . The cisLsZIP gene was distributed among the 8 chromosomes of lettuce. Chromosome 4 contains most the ZIP genes, while chromosome 9 contains no LsZIP genes. Chromosome1, chromosome3 and chromosome6 have two genes, chromosome3 and chromosome8 have three genes, chromosome2 and one gene in chromosome7. There was no positive correlation between chromosome length and the number of ZIP genes. After calculating the tandem replication events in the LsZIP gene family of lettuce by blastN, four tandem repeat genes (LsZIP4/6 and LsZIP7/11) were found. Two pairs of tandem repeat genes are located on chromosomes 3 and 4. Although LsZIP10 and LsZIP19 are also located on chromosome 4 and the distance between them is very small, surprisingly there is no tandem replication event between them. In addition to the tandem repetitive events, MCScanX methods identified 5 segmental repetitive events, containing 4 ZIP genes on nine chromosomes of lettuce. Several repetitive segmental events did not occur in the LsZIP gene family, such as LsZIP8-LAST_5X122561/LAST_3X58221, LsZIP12- LAST_8X48901. The gene protein sequences of four segmented events overlapping with the lettuce ZIP gene family were submitted to HMMER for searching, and no specific domains were found. These results suggest that some LsZIP genes may have evolved by gene duplication and that fragment duplication events are one of the driving forces of LsZIP gene evolution.As shown in LsZIP gene family, we constructed a comparative synonymous map of lettuce related to six different species, including Arabidopsis, cabbage, Cucumber, Tomato, Soybean and Sunflower. Monocotyledonous plants  were not used. The results are shown in Figs LsZIP genes a syntenic relationship, 15 soybean genes have syntenic relationship with 7 LsZIP genes. In sunflower, 10 genes have a syntenic relationship with 8 LsZIP genes. In cabbage, 7 genes have shown syntenic relationship with 6 LsZIP genes. In tomato, cucumber and Arabidopsis, 6 genes have syntenic relationship with 6 LsZIP genes. The numbers of relationship with 6 LsZIP genes. Pairs between the other six species  were 902, 569, 499, 450, 411 and 354. The LsZIP3 and LsZIP18 genes are syntenically relationship in all six species, suggesting that these orthologous pairs may share a common ancestor. Some LsZIP genes were found to be associated with two or three syntenic gene pairs . These genes are thought to have played a crucial role in evolution. It is interesting to note the lettuce LsZIP gene occurs in most syntenic relationships with soybean and not with sunflower in the composites. However, sunflower has a syntenic relationship with most of the ZIP genes of lettuce. In addition, the LsZIP1 gene has been identified as a collinear gene pair between lettuce and cucumber/soybean/tomato/cabbage but not in Arabidopsis/sunflower, and the LsZIP2/LsZIP6 genes are unique collinear gene pairs of lettuce and sunflower. This may could indicate differences between these direct pairs and different species.To further infer the phylogenetic mechanisms of the lettuce ZIP gene family, the Ka/Ks ratio of all ZIP genes was calculated as shown as ZIP genes, including all segmental and tandem duplicated ZIP genes, are all less than 1, indicating that purifying selection has played an important role in the evolution of these synteny genes.To better understand the evolutionary constraints acting on the LsZIP genes were induced to different events. In the Zn-excess treatment, four LsZIP genes were most highly expressed at 12 h, fifteen LsZIP genes were most highly expressed at 24 h, and the relative expression of one LsZIP gene was lower than that of the control; in the Zn-deficiency treatment, six LsZIP genes were most highly expressed at 12 h, and fourteen LsZIP genes were most highly expressed at 24 h. In Fe-excess treatment, four LsZIP genes had the highest expression at 12h, fifteen LsZIP genes had the highest expression at 24h, and the relative expression of one LsZIP gene was lower than the control; in Zn-deficiency treatment, two LsZIP genes had the highest expression at 12h, and fourteen LsZIP genes had the highest expression at 24h. The relative expression of four LsZIP genes was lower than that of the control. It is worth noting that most of these LsZIP genes are up-regulated under each element stress, indicating that they are actively involved in element transport. At the same, LsZIP genes also vary in their response to different element stresses. For example, LsZIP17 (Group Ia) was dramatically induced more than 16-fold under Zn-deficiency; LsZIP19 (group Ia) was dramatically induced by more than 11-fold under Zn-excess, but under Fe-excess and Cd stress, the change in expression of LsZIP17 was only 1.62-fold and 2.54-fold, respectively; LsZIP19 still has 6.31-fold and 7.25-fold expression. Under Fe-excess stress, the expression of LsZIP12 and LsZIP4 (group Ib) was significantly up-regulated more than 12-fold and 23-fold, LsZIP12 was also dramatically increased by 29-fold under Fe-deficiency, but the gene expression of LsZIP4 was significantly down-regulated. Under Cd stress, the expression levels of LsZIP12 and LsZIP4 increased 5.18- fold and 6.21-fold, respectively. LsZIP1, LsZIP3 and LsZIP13 (group Ic) were upregulated under all element stresses. LsZIP15 and LsZIP16 (group II) were up-regulated under Zn-deficiency, Zn-excess and Fe-excess. However, the expression of LsZIP15 was up-regulated 13.11-fold under Fe-excess stress. Under Fe-deficiency stress and Cd stress, the expression of the genes is slightly up-regulated. This indicates the important role of LsZIP15 in the response to Fe-excess stress. LsZIP14, LsZIP18 and LsZIP20 (group III) showed similar expression patterns as LsZIP15 and LsZIP16, but they have the highest gene expression under Zn stress, and they were actually not hypersensitive to Cd stress.To confirm that they are involved in the transfer of elements and their expression in the transfer of elements, the expression of 20 genes from five groups under Zn-deficiency, Zn-excess, Fe-deficiency, Fe-excess and Cd stresses were validated by qRT—PCR. The results of qRT-PCR analysis showed that under the stress of different elements, 20 ZIP gene family in plants such as Arabidopsis and rice. In addition to these genes playing a vital role in the transport of Zn and Fe, leaves are also involved in the transport of heavy metals such as Cd. However, there are no studies on the function and gene expression of LsZIP under elemental stress in lettuce. Therefore, this study aims to analyze the LsZIP gene family in the hope of investigating its possible effects on the transport of elements in lettuce and the accumulation of Cd. The whole genome sequence of lettuce allows researchers to identify and characterize different gene families in lettuce [ZIP genes are mainly focused on the transport and uptake of Zn and Fe, and there are few reports on the response to Cd, especially in leafy vegetables. As a leafy vegetable grown worldwide, lettuce has a high uptake capacity for Cd, but there are no reports on the identification of the ZIP gene in lettuce. In this study, based on previous research and the study of LsZIPs characteristics in other plants, we identified a total of 20 LsZIPs by genome-wide identification. Then, the gene structure, chemical and physical properties, phylogenetic relationships, conservative motifs, cis-regulatory element composition, chromosome location, synlinearity analysis and expression patterns under different element stresses were studied. The results provide valuable and important information to better explore the function and evolutionary relationship of these LsZIPs.Previous studies have identified the  lettuce . HoweverZIP gene members were identified by searching the lettuce genome. According to the Evalue obtained by hmmsearch, these members are named LsZIP1 to LsZIP20. The LsZIP genes usually have 1–3 exons, and the number of exons in the same subfamily is very similar. The LsZIP gene in the group III contains the largest number of exons. The LsZIP genes in group Ib and group II have conservative gene structure, suggesting that are closely related evolutionarily. Interestingly, the LsZIP gene in group III contains abundant short exons. Xu [LsZIP gene family.In our study, a total of 20 xons. Xu  hypothesLsZIP genes are distributed on chromosomes 1–8 of lettuce, whereas chromosome 9 does not contain LsZIP genes. In addition, the six LsZIP genes were distributed on chromosome 4, more than on the other chromosomes. The appearance of gene family members is usually accompanied by gene duplication and leads to new functions in the evolution of plant genomes. In our study, only two pairs of LsZIP genes were identified as segmental duplications. However, in LsZIP8 and LsZIP12, segmental duplications were identified as genes outside the LsZIP gene family, even if they did not contain specific domains. Among them, LsZIP8 was identified as segmental duplications with two genes. Syntenic LsZIPs were located at the beginning or end of chromosomes. In addition, there were three pairs of LsZIP genes on chromosomes, which are very close and closely related to each other. This implies that they may have a similar origin. Therefore, to further study the evolutionary relationship of this gene family, it is of great important to study their repetitive relationship.According to chromosomal mapping analysis, LsZIP gene family contains cis-acting elements that respond to hormones and stress, such as methyl jasmonate, abscisic acid, auxin, and salicylic acid. In a previous study, Akther [ZIP gene expression; furthermore, Kabir [ZIP genes of sunflower were regulated in response to iron deficiency; Matsuoka [AtZIP1 expression under iron sufficiency or iron deficiency conditions. This suggests that LsZIP gene expression may be induced by several plant hormones, such as salicylic acid, MeJA, abscisic acid, gibberellin, and auxin. Methyl jasmonate is an important hormone in plants. In the presence of insect pests, methyl jasmonate can serve as a key signaling factor of plant resistance to biological invasion [LsZIP gene. This suggests that the LsZIP gene family may regulate plant growth through hormone signaling pathways to adapt to elemental stress.After further analysis of the motifs, it was revealed that there are very similar motifs in groups I and II and their subgroups, which indicates that they are very conservative in evolution. No similar motif was found in group III. The , Akther  found th, Akther  found the, Kabir  found thMatsuoka  found thinvasion . In addiinvasion . Auxin iinvasion . Similarinvasion . Abscisiinvasion , therebyinvasion . In addiZIP family is not only responsible for the transport of Zn and Fe in plants, but also other divalent metal ions, including some heavy metal ions. For example, BcZIP2 can transport Zn2+, Fe2+, Cd2+ and Mn2+, and under Hydrogen-rich water treatment, the relative expression levels of BcZIP2 and BcZI42 are significantly down-regulated, and the Cd content in pakchoi is also reduced [AtZIP2 and AtZIP4 were identified as involved in copper transport [SlZIP4 gene in tomato increased, and shwed a concentration-dependent increase with increasing Cd concentration. A similar situation was also observed in rice, where the relative expression of the OsZIP4 was increased gene under Cd stress [LsZIP gene in leaves of lettuce treated with Zn-deficiency, Zn-excess, Fe-deficiency, Fe-excess and Cd for 12, 24, and 48 hours. Our results showed that most of the 20 LsZIPs were up-regulated under Zn treatment, Fe treatment and Cd stress, LsZIP genes were mainly expressed at 12 h and 24 h. Most of the LsZIP genes reached their highest expression level at 24 h and responded most strongly to elemental stress, but the extent of the up-regulation was differed among the different stress treatments. For example, LsZIP4 was significantly up-regulated 23.44-folds under Fe-excess treated, but gene expression was significantly down-regulated under Fe-deficient treatment. LsZIP12 was significantly up-regulated 12.3-fold and 29.8-fold under Fe-excess and Fe-deficient treatment. But not sensitive to Zn treatment. The same phenomenon also occurred in LsZIP17. Its gene expression was significantly down-regulated under Zn-excess and Fe-deficient treatments, but it was significantly up-regulated by 16.93-fold in Zn-deficiency. This indicates that different LsZIPs have different roles in the transport of elements in lettuce and show a tendency toward the elements. Interestingly, the relative expression level of many LsZIP genes increased under Cd treatment. For example, the expression level of LsZIP10 increased by 9.26-flods. Compared with the group I, the expression levels of group II and group III under Cd stress increased less. Although there are reports of the involvement of ZIP genes in Cd transport in Arabidopsis and tobacco [LsZIP genes in lettuce in response to Cd stress is far greater than these, which may explain why lettuce has a high accumulation and transport capacity for Cd.The  reduced . After uransport . Under Cd stress . In this tobacco , 58, theZIP gene family was mainly focused on the transport of Zn and Fe [ZIP gene family may play a crucial role in regulating the transport of Zn and Cd. For example, in rice, OsZIP1 can excrete Zn and Cd to prevent excessive accumulation [ZIP genes [In previous studies, the n and Fe , 10 and n and Fe , while tn and Fe , but stun and Fe . The ZIPmulation . StudiesIP genes . LettuceZIP genes in the lettuce genome. We investigated their physiochemical properties, cis-regulatory elements, motif composition, gene structures and phylogenetic relationships. Collinearity analysis and chromosomal mapping revealed the duplication relationship of lettuce LsZIPs. These LsZIPs were divided into three groups distributed on 8 chromosomes and comprising 1–7 exons. Phylogenetic analysis showed that the ZIP proteins of lettuce were divided into three types and five LsZIP genes were closely related to monocots. Synonymous analysis showed that two LsZIP genes were duplicated by segmental. The LsZIP gene was found to have a variety of cis-acting elements related to hormones and anti-stress response factors. Finally, expression analysis showed that the relative expression of the ZIP gene after treatment was generally higher at 12h and 24h, and decreased at 48h. The relative expression of 18 ZIP genes increased significantly with Cd stress. Overall, this study provides insights into the potential functional role of the ZIP gene in lettuce. Comprehensive analysis will help select candidate LsZIP genes for further functional characterization. In the next study, three candidate genes  with high expression under Cd stress will be selected to further verify their functions.In the current study, we performed an analysis of 20 S1 FigIdentical amino acids are indicated with red shading (100%) and similar amino acids are indicated with yellow (80%) and green (60%) shading. The seven domains are shown as a red line above the sequences.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TableLs’ indicating the lettuce species abbreviated from L. sativa; b: NCBI database unique digital identifier for the gene; c: Lo-cations represent the coding region of the gene. can estimate of the stability of your protein in a test tube, a protein whose instability index is smaller than 40 is predicted as stable, a value above 40 predicts that the protein may be unstable.Note: a: Name of gene model was modified from the annotation of the lettuce genome v7 (from NCBI), the prefix ‘(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


P)av of the compound. Moreover, the average of the final particle sizes P)av. These findings are useful for predicting the size of nanodrugs prepared through the antisolvent precipitation method.The aim of the present study was to systematically examine the effects of variations in the process parameters of the antisolvent precipitation method employed in the preparation of excipient-free pure nanoparticles of five existing/potential psychotropic drugs, namely amitriptyline hydrochloride (AMI), coumarin 6 (COU), curcumin (CUR), nortriptyline hydrochloride (NOR), and prochlorperazine dimaleate (PRO). In the preparation protocols employed, AMI and NOR were expected to be charged enough to be identified as surface-active molecules. Through the employment of five different preparation protocols, the effects of varying the flow rate, the compound concentration in the solvent solution  Psychiatric disorders represent an increasing health burden on an international level and are often associated with a great socio-economical and emotional burden for patients and caregivers. Worldwide, around 450 million people have experienced suffering due to psychiatric disorders, with depression (300 million), bipolar affective disorder (30 million), and schizophrenia 23 million) being the most common conditions . Accordi3 millionBy downsizing the drug particles, a much larger effective surface area could be achieved and the accompanied increased dissolution rate and saturated solubility may improve the bioavailability of drugs ,5. The bAmong all of the nanoparticulate drug delivery systems, those based on polymers and lipids  represent a significant portion of the drug delivery systems that reach clinical trials . HoweverThe preparation of nanoparticles in the absence of excipients can be broadly classified into two approaches, namely top-down and bottom-up. The top-down approach focuses on the size-reduction of bigger particles to nanosized particles . Essentially, every drug can be milled to smaller particles regardless of their solubility in aqueous or non-aqueous solvents . It shouIn the bottom-up approach, nanoparticles are generated from drug molecules in solution using, e.g., the antisolvent precipitation method or evaporative precipitation technique ,20. ThisThus, in the present study, we employed one of the methods following the bottom-up approach, namely the antisolvent precipitation method. Nanoparticles were created for a number of potential and existing psychotropic drugs, namely curcumin (CUR), coumarin 6 (COU), nortriptyline hydrochloride (NOR), amitriptyline hydrochloride (AMI), and prochlorperazine dimaleate (PRO). AMI and NOR are well-known antidepressants. PRO is used in the symptomatic management of psychotic disorders and short-term management of nonpsychotic anxiety, while CUR exhibits significant antidepressant effects, possibly through the inhibition of monoamine oxidase A and B enzymes , and somAnalysis of the final size of the particles olecules . It is oThe principle behind the antisolvent precipitation method is to exploit the solubility of a drug in different miscible solvents, one good solvent and one bad solvent (antisolvent). First, the drug is dissolved in the solvent and, afterwards, is quickly mixed with the antisolvent. The fast diffusion of the drug solvent solution into the antisolvent causes a high supersaturation, which is the driving force for the precipitation . The degition C* ,22,26,27The precipitation process consists of several steps: (i) nucleation, (ii) particle growth, and (iii) agglomeration. Classical nucleation theory describes the initial step of the crystallization as the spontaneous assembly of a few molecules, which creates a thin interface between a solid and a liquid phase . Energy ribed as .(2)ΔG*=The kinetic parameter describing the rate of nucleation erations :(4)J=AneThe parameters enerated . Increasin value . A furthAfter the formation of stable nuclei, particle growth happens through the condensation of molecules onto an already growing surface, thus reducing the supersaturation in the system . The parDuring agglomeration and aggregation, the number of particles decreases and the particle size increases. The driving force behind all three stages of the precipitation process is supersaturation. Alteration of the parameters affecting supersaturation can therefore be used to design particles with desirable sizes. After particles are made, several destabilization routes, such as Ostwald ripening, may lead to further particle growth. Immediate drying, e.g., spray drying or freeze-drying, may provide prolonged stabilization .Curcumin was received as a gift from Capital Medical University in China. Coumarin 6 , nortriptyline hydrochloride , amitriptyline hydrochloride , prochlorperazine dimaleate (powder), and sodium hydroxide  were purchased from Sigma-Aldrich, Steinheim, Germany. Absolute ethanol , acetone , and distilled water were used in the experiments. Standard buffer solutions of pH = 4.008, pH = 7.413, and pH = 11.00 were purchased from Riedel-de Haën, Steinheim, Germany.Solvent solutions with saturation concentration (Cs) and half of the saturation concentration (½Cs) of CUR, COU, NOR, AMI, and PRO were prepared and subsequently filtered through a 0.2-µm nylon syringe filter . The solvent solution of the compound (10 mL) was added to the antisolvent (100–200 mL) in portions of 1 mL at a fixed flow rate (1 or 2 mL/min) under the stirring speed of 1000 rpm . A sample was removed after each addition. The samples were centrifuged at 20 °C and 3000 rpm . The dynamic light scattering (DLS) data presented correspond to samples that were prepared by the re-dispersion of the centrifuged particles in antisolvent solutions, where a volume (950 µL) of the upper layer was removed for analysis.The concentration of the compound in the solvent solution  was used for evaluating the morphology of the particles and confirming the particle size using secondary electron detectors (inLens and SE). Samples prepared by the solvent-evaporation or freeze-drying of the compound particles were put on carbon tape and coated with iridium . For solvent-evaporation, droplets of compound re-dispersions were put on microscopy slides and allowed to dry at room temperature. Afterwards, these samples were coated with carbon .The zeta potential (ZP) of the re-dispersions (in antisolvent) of the particles of the end dispersions of protocol No. 1 was measured using the Zetasizer. The measurements were carried out at 25 °C with disposable folded capillary cells  using the Zetasizer and Smoluchowski approximation. The value of pH was also measured with a 914 pH/Conductometer  after calibration with buffers with pH = 4.008, pH = 7.413, and pH = 11.00.The crystal structures of freeze-dried compound particles were determined with a Bruker D8 Advance diffractometer  using a spinning flat plate configuration.For preparation protocols No. 1–No. 5 , the resnm (PRO) . The valsiolvent . For thesiolvent . Exceptisiolvent , and (iisiolvent .The results of the measurements of ZP and pH of the re-dispersions (in antisolvent) of the particles of the end dispersions of protocol No. 1 are presented in The morphology of the produced particles was observed by SEM. All of the particles that were dried by the evaporation of solvent were spherical in their shape , where tThe X-ray powder diffraction (XRD) patterns for COU, CUR, AMI, NOR, and PRO can be found in Based on the XRD patterns, all of the compounds gave crystalline particles. CUR has been reported to crystallize in two different polymorph forms, namely form 1 and form 2 . ObtaineThe spherical shape of the particles was confirmed through inspection of the SEM images obtained for solvent-evaporated samples a,c,e,g,ianalysis . Evidentanalysis  correlatanalysis .Surface-active properties have been reported for AMI , NOR 2525, and PKnowing the pH of the antisolvent , the valIn the following, the effects of various parameters, including the flow rate, Flow rate usually has a minor impact on the particle size, because it exerts two opposing effects with respect to supersaturation ,48,49,50Our results show that increasing the flow rate from 1 mL/min to 2 mL/min  b. This o(I) On the one hand, a higher encing J .(II) On the other hand, a higher In our experiments, increasing the SAS-ratio from 1:10 to 1:20 could induce the formation of smaller or larger particles (or particles with little change in their size) c. This hSometimes, changing a process parameter could induce a very large change in ompounds a–c. In p0solvent b, and th0solvent c. For AMFor studying the effect of pH of the antisolvent on the particle size, we selected AMI d, where ion (1)) . In the For bases (B) that are much less soluble in water than their corresponding salts , the reressions .(7)CB*,4 ± 1 °C ), the va−2 M and its aggregation number is 7 . The suInspection of the data of For COU, CUR, and PRO particles, the concentration of the compound in the solvent solution varied in the range nt<10 mM . This me(logP)av a. We sugAs discussed before in nt<80 mM , which wller DHf b. FurtheThe antisolvent precipitation method was successfully employed in the preparation of excipient-free, pure nanoparticles of amitriptyline hydrochloride (AMI), coumarin 6 (COU), curcumin (CUR), nortriptyline hydrochloride (NOR), and prochlorperazine dimaleate (PRO) using five different preparation protocols . The fin


Background: We evaluated a strategy to shorten the time from admission to surgery in patients with proximal femur fractures on chronic antiplatelet therapy. We reported a 12-month follow-up on complications and quality of life (QoL). Methods: Multicentre, open-label, randomized, parallel clinical trial. Patients were randomized to either early platelet function-guided surgery  or delayed surgery (control group). Medical and surgical complications and QoL (EQ-5D-5L questionnaire) were assessed during the hospital stay, and after hospital discharge at 30 days, and 6 and 12 months. Results: From 156 randomized patients, 143 patients underwent surgery. The mean age was 85.5 (7.8) years and 68.0% were female. After hospital discharge, 5.7% of patients had surgical wound complications and 55.9% had medical complications, with 42.7% having serious adverse events. QoL improved significantly after surgery, with the best scores at the six-month follow-up. The overall mortality was 32.2%. There were no differences between early and delayed surgery groups in any assessed outcomes. Conclusion: It seems safe to reduce the time of surgery under neuraxial anaesthesia in patients with hip fractures on chronic antiplatelet therapy by platelet function testing. QoL in particular improves in the first six months after surgery. In developed countries, the incidence of proximal femur fractures in the elderly population has increased. This group of patients had chronic underlying pathologies, mainly of cardiovascular origin with chronic anti-platelet drug use. Associated comorbidities and chronic treatments increase perioperative complications in elderly patients, especially those undergoing proximal femur fracture surgery.Early surgery within 24 to 48 h) has been reported to have a positive effect on morbidity, hospital stay, perioperative complications, and mortality associated with a proximal femoral fracture 4 to 48 h,6,7,8,9.Before performing hip fracture surgery, a complete assessment of the patient must be made. This includes regular medication revision, stabilization of the patient’s pathologies, and choosing an anaesthetic technique that will be most beneficial to the patient. At the moment there is little evidence on the best type of anaesthesia that would reduce perioperative and long-term complications in patients on chronic antiplatelet therapy ,14,15,16As an alternative, we developed a clinical trial that evaluated an individualized strategy according to the platelet function test to shorten the time from admission to surgery without increasing perioperative complications in elderly patients with proximal femur fracture on chronic antiplatelet therapy ,33. ThisWe followed the recommendations of the Patient-Reported Outcomes (PRO) extension of the CONSORT guide for reporting this clinical trial .® .This is a multicentre, open-label, randomized, parallel clinical trial, performed in four Spanish hospitals. The inclusion criteria were: Adult patients over 18 years of age with a proximal femur fracture and treatment with chronic antiplatelet agents such as cyclooxygenase inhibitors  > 100 mg/day or triflusal > 300 mg/day) or P2Y12 receptor inhibitors . Patients underwent orthopaedic surgery with neuraxial anaesthesia. All patients signed the informed consent before participation. Patients were recruited between 26 September 2017, to 5 December 2020. Patients with multiple or pathological fractures, treatment with vitamin K antagonists or new oral anticoagulants, and congenital or acquired coagulopathy were excluded. After hospital discharge, the information was collected through telephone calls to the patients or caregivers and a review of medical records. The data was collected on the electronic database platform ClinapsisOn admission, antiplatelet medication was discontinued, and patients were randomized to early surgery  or delayed surgery (control group), in both cases with neuraxial anaesthesia.9/L of functional platelets, in which case, surgery was scheduled in the following 24 h. When the patient had <80 × 109/L of functional platelets, platelet function was measured daily to verify the opportune moment to schedule surgery. If the minimum platelet threshold was not reached on the third day, surgery was scheduled following the margin of safety established for each antiplatelet drug as specified in the control group.Experimental group: Platelet function was measured on admission to the emergency room to assess the anaesthetic safety of taking the patient to early surgery, having a minimum threshold of 80 × 10Control group: Surgery under neuraxial anaesthesia was performed following the safety margin of each antiplatelet agent, being 3 days for ASA > 100 mg/day and triflusal > 300 mg/day, 5 days for clopidogrel and ticagrelor, 7 days for prasugrel and 10 days for ticlopidine ,22,23,24Orthopaedic surgeons acted in accordance with their usual clinical practice. Surgical technique, transfusion, and perioperative antibiotic and antithrombotic medication followed the protocol of each hospital. Medical and surgical complications and quality of life (QoL) assessments were collected during hospital stay and after hospital discharge, at 30 days, 6 and 12 months. Among the surgical complications, the presence of infection, dehiscence and/or hematoma of the surgical wound, dislocation of the prosthesis and/or surgical reintervention were evaluated. A medical complication was defined as any change in health status that required treatment or hospital admission, such as infections, changes in mental status, cardiovascular disease and/or death. Serious adverse events were defined as any event that caused death, was life-threatening, required hospitalisation, prolonged existing hospitalisation, or caused permanent or significant disability.QoL was measured using the validated Spanish version of the generic questionnaire EQ-5D-5L (EuroQoL) , consistThe instrument was applied at six different times after the fracture: before surgery, at 5 days, 1, 6- and 12-months post-surgery, considering the basal value of the QoL before the fracture obtained at the time of enrolment. Follow-up was done face-to face during the hospital stay and by telephone after discharge. The questionnaire was answered by the patient or a relative/caregiver.The study was approved by the ethics committee of each participating centres and registered in clinicaltrials.gov NCT03231787). The protocol and perioperative results have already been published 31787. Th.Complications  and QoL were analysed per protocol, considering all randomized patients who underwent surgery. Mortality was analysed by the intention-to-treat (ITT), including all randomized patients.A descriptive and comparative analysis was made between the evaluated interventions of the medical and surgery-related events presented during the 12-month follow-up.The qualitative variables are presented as frequencies and percentages, and for the quantitative variables the mean with the standard deviation (SD) or the median and the IQR according to normality were calculated. To compare the results between groups, the Chi square test was applied.Mortality was reported as a cumulative incidence proportion. Survival was calculated from the time of hospital admission to 12 months after surgery by group. The survival differences were calculated by Kaplan-Meier method using the Log rank test.When there was no data prior to the fracture or surgery, the average of all the patients of the corresponding evaluation was imputed.If a measurement was missing between two points of the same patient, the measurements before and after the missing data were averaged.If tracking was lost, the last recorded value was forward dragged.When the patient died at the point of measurement, 0 was imputed and no further follow-ups were recorded.The analysis of QoL included all patients who underwent surgery and had preoperative QoL and at least one postoperative measurement. We imputed missing data with the following criteria:We evaluated QoL throughout the study, from baseline to the end of follow-up. For changes in the VAS and EQ-5D-5L scores, a two-way ANOVA was performed. The factors were group (intervention and control), time , and the interaction between them. The ANOVA was performed using a general linear model (GLM) procedure.We used a significance level of 0.05. All statistical analyses were performed with IBM Corp. Released 2017. IBM SPSS Statistics for Windows, Version 25.0.; IBM Corp., Armonk, NY, USA.n = 78) and control group (n = 78). Before surgery, one patient in the intervention group and two in the control group removed their consent; three patients in the control group were withdrawn for not meeting some inclusion criteria. This report corresponds to the remaining 150 patients; 77 participated in the experimental group and 73 in the control group  and a median of appearance of 110 days (from 1 to 350). In total, eight (5.7%) patients presented surgical wound complications, 80 (55.9%) presented medical complications, and 61 (42.7%) presented serious adverse events without differences between groups. The most common complications after discharge were death (34 patients), urinary tract infection (24 patients) and cardiovascular diseases (13 patients) .n = 48). The mean survival time was 275.9 days (11.6) with no statistical difference between groups (p = 0.561) (n = 34) with no difference between groups (p = 0.521).In an ITT analysis, the overall mortality of the study was 32.2% (= 0.561) . Post-dip < 0.0001) but without reaching baseline values and without differences between groups  of the 143 patients that underwent surgery were included. The global assessment of quality of life measured with the VAS prior to the fracture was 63.4 and 39.0 after the fracture at hospital admission. In successive follow-ups after surgery, VAS improved significantly (n groups .p < 0.0001), with no difference between groups (The QoL index evaluated with the EQ-5D-5L instrument had an average of 0.555 before the fracture and decreased to −0.349 before surgery. After surgery, patients had a progressive and significant increase in QoL index scores, reaching up to 0.340 at 12-month follow-up (n groups .Accelerating the time to surgery with neuraxial anesthesia in patients with a proximal femur fracture chronically medicated with antiplatelet drugs did not result in significant differences in quality of life nor in the incidence of complications when compared with those receiving delayed surgery at 12-month follow-up.The chronic use of antiplatelet agents is an important risk factor for complications such as bleeding and mortality in patients undergoing surgery. Therefore, it is recommended to suspend the medication prior to the surgical procedure, especially if neuraxial anesthesia is used due to its high risk of bleeding ,23,38. HThe average age of around 80 years is consistent with global reports on the incidence of proximal femur fracture in patients with cardiovascular comorbidity and a higher frequency in women ,42,43,44Most complications in our study occurred during the first 4 months after surgery, with no differences between compared groups. The most frequent postoperative medical complications were urinary tract infections and cardiovascular events, which occurred after 60 and 100 days from randomization, respectively. These complications are commonly reported in the literature ,43,46,50Quality of life has been under investigated in patients with proximal femur fracture and chronic antiplatelet therapy. This study found that, after surgery, the general assessment of health status and the patient’s index of quality of life improved significantly with respect to the moment of the fracture, especially in the first six months after the intervention; however, they did not reach the pre-trauma baseline values. This is consistent with various studies on quality of life in proximal femur fractures . The subThis study stands out for its design, as it was a multicentre randomized clinical trial with prolonged patient follow-up. It contrasts with most published primary studies on complications and quality of life that are observational and retrospective. However, the relatively small sample size has been a limitation. Studies with a larger number of patients would be required to corroborate the findings of this study.This study suggests that it is possible to reduce the time to surgery under neuraxial anaesthesia in patients with hip fracture receiving chronic antiplatelet therapy using a platelet function test, without increasing post-surgical complications, serious adverse events, or mortality, and not worsening their quality of life up to 12 months after surgery.


Autism spectrum disorder (ASD) is a widespread developmental disorder of the nervous system with an unclear etiology and pathogenesis. Its global incidence is currently increasing, and no effective drugs are available to improve its core symptoms. Nonpharmaceutical therapy can effectively relieve the core symptoms of autism, has fewer side effects than drugs, and is easily accepted by patients. This systematic and network meta-analysis aimed to evaluate the impact of non-pharmaceutical therapy on autism to explore preferable therapeutic options for autism.Online databases, including the China National Knowledge Infrastructure [CNKI], SinoMed, Wanfang Database [WF], China Science and Technology Journal Database [VIP], MEDLINE, Web of Science, EMBASE, and the Cochrane Library will be searched for randomized controlled trials of nonpharmacological interventions for autism published before October 2021. Two researchers will be independently responsible for the literature screening, data extraction, and quality assessment. Standard paired and Bayesian network meta-analyses will be performed using RevMan 5.3 Software and GEMTC 0.14.3, to compare the efficacy and safety of different nonpharmacological regimens.The results of this systematic and network meta-analysis will be submitted to a peer-reviewed journal for publication.This study provides a comprehensive and reliable evidence-based reference for the efficacy and safety of different non-pharmacological interventions for autism.CRD 42021275571 The prevalence of ASD among 8-year-old children in the United States increased from 6.7% in 2000 to 14.6% in 2012,Autism spectrum disorder (ASD) is a serious developmental disorder of the nervous system with core symptoms of social dysfunction, communication barriers, narrow interests, and rigid behaviors.,11 causi[–18] Medications such as antidepressants, stimulants, antipsychotics, alpha agonists, and anticonvulsants are often used to relieve anxiety, ADHD symptoms, compulsions, and other repetitive behaviors, mood lability, irritability, aggression, and sleep disturbance. However, these drugs cannot assuage the core symptoms of communication skills and stereotyped behaviors and can induce multiple side effects such as sedation, increased appetite and weight gain, disrupted sleep, prolactin elevation, and extrapyramidal symptoms.[–21]The current treatment for autism is mainly behavioral interventions combined with drug therapy.–18 Medicsymptoms.–21[–26] Given the lack of reported adverse reactions or interactions associated with non-pharmacological therapies, this treatment is more easily accepted by patients than prescription drugs. However, current meta-analyses have only compared the effectiveness of a single non-drug therapy. A network meta-analysis is necessary to evaluate the impact of different types of non-drug treatment on the core symptoms of patients with ASD and to explore the best non-drug options.Non-pharmacological treatments, such as yoga, acupuncture, massage, music therapy, and manual therapy, are simple and economical methods that can effectively alleviate the core symptoms of ASD, such as social barriers, communication barriers, and stereotyped behaviors.–26 GivenThis systematic review and network meta-analysis aimed to evaluate the potential safety and effectiveness of different types of nonpharmacological interventions for ASD.22.1This systematic review and network meta-analysis aimed to evaluate the efficacy and safety of different nonpharmaceutical interventions for ASD.This study will be reported with the Preferred Reporting Items for Systematic Review and Meta-Analyses statement.2.22.2.1Regardless of the language or publication status, only clinical randomized controlled trials (RCTs) of nonpharmacological treatment for children with autism will be included. Repeat published studies, non-RCTs, and laboratory studies were excluded.2.2.2Participants were adolescents between the ages of 3 and 17 years who met the diagnostic criteria for ASD in the Diagnostic Statistical Manual or the International Classification of Diseases, regardless of sex, race, study area, or educational status.2.2.3Interventions included any type of clinically performed non-pharmaceutical therapy for patients with autism, such as music therapy, play therapy, applied behavior analysis, TEACCH training, relationship development intervention, manual therapy, massage, acupuncture, and yoga.Control: no invention or treatment other than non-pharmaceutical therapy .2.2.4Primary outcomes included changes in the core syndromes  of autism. Evaluation will be performed using the symptom scores of the Childhood Autism Rating Scale (CARS) and the Autism Rating Scale (ABC). The CARS is one of the most widely used autism rating scales that is suitable for children over 2 years of age, has good reliability and validity, and can distinguish autism from retardation and judge its severity. ABS has 57 items of behavioral characteristics in 5 aspects, including sensory, relating, body and object use, language, and social and self-help, scored by parents or teachers for children over 2 years old. The scale is scored “0” to “158” points, with points over “67” indicating a diagnosis of autism.Secondary outcomes included the autism diagnostic observation scale, autism diagnostic interview-revised, social responsiveness scale, clinical global impression scale, social interaction questionnaire, Child Behavior Checklist, and Wexner Rating Scale.2.3Eight electronic databases, including 4 English databases  and 4 Chinese databases , will be searched for eligible studies published before October 31, 2021, regardless of language and publication type. Different strategies will be applied to electronic databases based on treatment terms , disease terms , and RCT terms  , both masters of (non)acupuncture and moxibustion, will monitor and evaluate the screening process. Two reviewers screened articles that met the inclusion criteria according to the title and abstract content. After the screening, another reviewer reviewed the articles. Disagreements between the 2 authors will be resolved through consensus or discussion with a third author (W. X.Q.). The systematic evaluation plan and full text will be elaborated upon during the selection. The main process consists of the following steps  and verified by a third party, and all disagreements were resolved through consensus. Data related to study identification , study characteristics , inclusion criteria , intervention type , and other factors that may introduce errors  will be included. If the information is clearly incorrect or incomplete, the first author will be contacted via email. Document was deleted when no response was received.2.5The risk of bias for the included studies will be independently assessed by 2 evaluators using the Cochrane Risk of Bias Tool,2.62.6.1I2 tests were used to detect heterogeneity in the included studies. If I2 < 50% and P > .10, then the study has no statistical heterogeneity or less heterogeneity, and the fixed effects model can be selected; otherwise, the random effects model can be used.Data analysis and paired meta-analysis will be performed using the RevMan software . Relative risk and standardized mean difference were selected as expressions of effect size for dichotomous and continuous variables, with 95% confidence intervals (95% CI). The Q and 2.6.2Owing to the anticipated heterogeneity, NMA within the Bayes framework will be performed with GEMTC 0.14.3 software based on a random effects model of indirect comparison results. The model is calculated using the Markov chain Monte Carlo algorithm.2.6.3Funnel plots will be used to assess the possibility of a small-sample publication bias when 10 or more studies are included. An asymmetrical, non-inverted funnel shape indicated possible publication bias. This may be related to the small sample size, hidden distribution, and inadequate implementation of the blind method.2.6.4In the case of heterogeneous results, a subgroup analysis will be conducted. In the presence of more than 2 variables, meta-regression was performed. A sensitivity analysis was performed to determine data reliability based on missing data, sample size, and heterogeneity.2.6.5Evidence quality will be evaluated by 2 independent authors in accordance with the Grading of Recommendations Assessment Development and Evaluation (GRADE) and classified as follows3Autism causes serious damage to the physical and mental health of children owing to the refractory and lifelong nature of its core symptoms. RCTs have shown that non-pharmaceutical therapies can effectively alleviate the core symptoms of autism. Due to the lack of reported side effects, this treatment is easily accepted by patients. However, no comparison has been conducted among the different non-pharmacological interventions applied to alleviate the core symptoms of autism. This systematic review and network meta-analysis will compare the effects of multiple nonpharmacological interventions by combining direct and indirect data. The ranking probability of treatment effects will also be obtained within the Bayesian framework to comprehensively analyze different non-pharmacological interventions. All available evidence regarding the impact of these therapies on the core symptoms of autism will be examined.3.1No ethical review was required, because this study did not involve raw data collection. These results provide strong evidence for the treatment of autism using non-pharmacological options. These findings will help clinicians make treatment decisions for autism. This systematic review will be published in a peer-reviewed journal and at an international conference.3.2This is the first systematic review and network meta-analysis of the efficacy and safety of nonpharmacological therapies for the relief of autism core symptoms. The results will reveal the advantages and disadvantages of various non-pharmaceutical treatment methods for autism and provide a basis for their clinical application to improve the quality of life of patients. Only studies published in English and Chinese were included because of language limitations; this phenomenon may lead to selection bias. In addition, the age and region of the patients and the quality of the literature may increase the possibility of heterogeneity. The methodology and quality assessment will follow systematic evaluation guidelines and standards to minimize the risk of bias.Conceptualization: Xin Jiang, Weixun QinData curation: Jiang Xiao, Xiaoqing Xu.Data extraction: Jiang Xiao, Xiaoqing XuFunding acquisition: Qing Yuan.Investigation: Song Min, Qing Yuan, Min Song.Methodology: Xin Jiang, Weixun Qin, Song MinProject administration: Qing YuanResources: Weixun Qin, Qing YuanSoftware: Xin Jiang, Min Song.Writing – original draft: Xin Jiang, Min Song, Weixun QinWriting – review & editing: Jiang Xiao, Qing Yuan


Rhodopseudomonas is known for its versatile metabolic capabilities and has been proposed to be used in a wide variety of innovative applications, ranging from biohydrogen and electricity production, bioremediation and as biostimulant in agriculture. Here, we report the isolation, characterization and genome sequence analysis of a novel Rhodopseudomonas species, strain HC1, isolated from the Hell Creek urban native restoration area. Whole genome-based analysis, average nucleotide identity (ANI) comparison, and growth characteristics identified this isolate as a new species of the Rhodopseudomonas genus, for which we propose the name Rhodopseudomonas infernalis sp. nov. Besides containing several nitrogenases for nitrogen fixation and hydrogen production, the HC1 genome encodes a unique gene cluster, not found in any other Rhodopseudomonas species, which encodes genes for the degradation of yet-unidentified aromatic PCB-type chemicals with potentially interesting biotechnological applications. The genomic features of Rps. infernalis HC1 indicate that it plays a positive role in the degradation of anthropogenic substances and aids the restoration of the Hell Creek watershed by contributing to N2 and carbon fixation and plant growth; however, the genome also contains several antibiotic resistance genes, indicating a broad range of antibiotic resistance in this environmental isolate.The genus  Bradyrhizobiaceae [Rhodopseudomonas isolate by selective cultivation in the lab. Besides the genotypic and phenotypic characterization of the new isolate, we also sequenced its whole genome and performed a whole genome-based taxonomic analysis. These analyses showed that the HC1 isolate should be classified as a new species of the genus Rhodopseudomonas.The Hell Creek watershed area is part of a nature restoration project around Hell Creek, situated in the suburban Omaha area. A collection pond has been constructed that collects rainwater and overflow runoff from a larger commercial and residential development area nearby, but is not directly connected to the creek itself. We had previously performed a metagenomic analysis of the watershed  and now obiaceae ; howeverRhodopseudomonas has been isolated from a wide variety of environments, ranging from aquatic sediments [Bradyrhizobiaceae and order Rhizobiales within the Alphaproteobacteria class [Rhodopseudomonas are Gram-negative, anoxygenic phototrophic bacteria and are characterized by a budding cell morphology and the presence of intracytoplasmic membrane system of the laminar type [Rhodopseudomonas belongs to the purple non-sulfur bacteria and grows anaerobically in the light or aerobically in the dark with a variety of different carbon sources and electron donors. However, Rhodopseudomonas has the exceptional ability to grow using the four major modes of metabolism, photoautotrophic, photoheterotrophic, chemoautotrophic and chemoheterotrophic, and is flexible in switching between these modes depending on environmental conditions [Rhodopseudomonas to optimize strategies for solar energy capture [ediments ,3, sludgediments ,5, soilsediments ,7, alkalediments  and eutrediments . The genia class . All Rhonar type . Rhodopsnditions ,12. Thisnditions ,14,15. I capture .Rhodopseudomonas perform nitrogen fixation using nitrogenase enzymes that convert atmospheric N2 gas into ammonia (NH3) and hydrogen (H2), thereby converting the nitrogen into a useable form for bacterial and plant biosynthesis. This same enzyme also provides Rhodopseudomonas the capability to produce large amounts of hydrogen (driven by ATP from light energy). Rhodopdseudomonas strains have also been studied extensively for their ability to degrade a wide variety of organic compounds [All ompounds ,18,19. Sompounds ,12,19.2 and produce hydrogen using light energy, and its ability of anaerobic degradation of aromatic compounds, Rhodopseudomonas has been proposed as an ideal bacterium for a variety of biotechnological applications, ranging from environmental bioremediation and biostimulation, to biofuel (biohydrogen) production and electricity production [Due to its metabolic diversity, the ability to capture Noduction ,13,20,21Rhodopseudomonas palustris was initially identified by van Niel in 1944 [Rhodopseudomonas genus: Rps. palustris, Rps. rutila, Rps. faecalis, Rps. pantothenatexigens, Rps. thermotolerans, Rps. rhenobascensis, and Rps. telluris [Rhodopseudomonas have been sequenced. As of to date, a total of 28 Rhodopseudomonas genomes were found in NCBI Genbank. Eight of them are from strains designated as Rps. rutila, two as Rps. palustris, four as Rps. faecalis, one as Rps. pantothenatexigens, Rps. thermotolerans, Rps. rhenobacensis, “Rps. boonkerdii”, and 10 of them have no species designation. With more genomic information becoming available on this genus in the last few years, it became clear that there was a need for taxonomic revision of the genus as several taxonomic discrepancies were described with new genomes being published [Rhodopseudomonas is widely distributed in soil and water environments it is frequently found in metagenomic analyses and correct taxonomic information is important to derive detailed conclusions from such broad metagenomic studies. Therefore, an updated phylogenetic analysis of the genus, based on whole genome comparisons and 16S rRNA comparisons, was published recently [Rhodopseudomonas palustris DSM 123 is the authentic type strain for that species, which had previously been debated, but previous confusion was shown to be due to errors in strain transfers over time. It also placed several of the previously misplaced strains into the Rps. rutila species group and confirmed Rps. rutila R1 as the type strain. in 1944 . There atelluris ,24,25. Sublished ,26. SincRhodopseudomonas infernalis sp. nov.A whole-genome-based comparison was performed with the new isolate HC1, and found the new isolate to be only distantly related to any of the currently described species. We now present the complete genomic and physiological characterization of this new species, for which we propose the name g) and resuspending the pellet in 1 mL of a methanol/acetone mixture (50/50). The suspension was sonicated for 2 min  using a microtip model FB120 Sonicator (Thermo Scientific) and centrifuged for 5 min at 5000× g. Spectra were collected from the supernatans using a Genesys 10S UV-vis spectrophotometer (Thermo Scientific).Microbial samples were isolated from Hell Creek in Omaha, Nebraska in April 2020, as described in Kyndt, 2020 . SamplesCells were observed using a MEIJI Techno  MT4200H microscope equipped with brightfield and phase contrast condenser, and images were obtained with an attached Motic digital camera .DNA was isolated from bacterial colonies grown under anaerobic light conditions, using the GeneJet DNA purification kit (Thermo Scientific). Purified DNA sample was analyzed for purity and concentration using a Nanodrop and Qubit, showing an absorbance 260/280 nm ratio of 1.7 with a concentration of 31.5 ng/µL. An amount of 300 ng purified gDNA was used for Illumina whole genome sequencing, and 1 μg was used for Oxford Nanopore long read sequencing.The Illumina DNA library was prepared following the Nextera DNA Flex library prep kit . Genomes were sequenced using 500 μL of a 1.8 pM library with an Illumina MiniSeq instrument. Paired-end (2 × 150 bp) sequencing generated 1,842,158 reads and 278.2 Mbps. Quality control of the reads was performed using FASTQC within BaseSpace , using a kmer size of 5 and contamination filtering. The data was assembled de novo using Unicycler  within PThe genome sequence was annotated using RAST  between whole genomes was calculated using JSpecies , which uThe single protein tree for protW, was generated by the multiple sequence alignment viewer within PATRIC , which uThe multiple sequence alignment for the 16S rRNA comparisons was performed using ClustalW . The evoa. Pigment extraction with MetOH/acetone revealed additional spectral peaks with maxima of 368 nm , and broad maxima at 498, 533, and 589 nm, indicative of the presence of spirilloxanthin-type carotenoids, as found in other Rhodopseudomonas species [Environmental water samples were collected from the northern pond at the Hell Creek watershed in 2020 as described earlier . A small species ,44,45.Rhodopseudomonas species, and chemotrophic growth appears to be preferred in this strain [Rhodopseudomonas strains tested, none of them were able to use L-arginine as the sole carbon source for heterotrophic growth [Strain HC1 was able to grow anaerobically in the light and aerobically in the dark; however, anaerobic light growth was very slow (color appeared after about 2–3 months of incubation). This anaerobic photosynthetic growth of the purified HC1 strain appears to be considerably slower than other s strain . The folc growth ,23,24. ARhodopseudomonas species [Rhodopseudomonas (68% of the identified reads). About 31% resulted in no hits from the database, which indicates that this is a species or strain that is not represented in the database.To identify the taxonomy of the isolated strain, we decided to sequence its whole genome. The whole genome sequencing provided a genome assembly with a genome length of 5,543,543 bp, in 43 contigs, and an average G+C content of 65.3%. The latter is close to the GC content of several of the other  species ,23,24. ARhodopsuedomonas genus [Rhodopseudomonas species was calculated using JSpecies , Rps. rutila R1 (81.6% ANI), Rps. faecalis JCM11668 (77.3% ANI), Rps. thermotolerans JA576 (82% ANI), and Rps. rhenobascensis (78.6% ANI). Therefore, strain HC1 should be regarded as a new species, different from the previously characterized Rhodopseudomonas species.Bacteria with ANI < 90% will have insertions and deletions of whole genes, they will have substantial base changes, and most proteins will have at least a few amino acid substitutions. In other words, they would be recognized in most cases as separate species. As can be seen in Rhodopseudomonas genomes, is consistent with the ANI analysis presented above ; however, these are on a separate clade and only have low ANI comparison values, indicating that HC1 belongs to a different species. The species rutila, faecalis, pentothenatexigens and thermotolerans are clearly more distantly separated on the whole genome tree, which is consistent with our earlier overview of the Rhodospeudomonas genus [A whole genome-based phylogenetic analysis of the ed above . The phyas genus .Rhodopseudomonas do not have genomes available yet, we also performed a 16S rRNA-based phylogenetic comparison to include all species in the comparison. The 16S rRNA sequences used were deducted from the whole genomes where available or directly obtained from NCBI Genbank. The resulting 16S rRNA phylogenetic tree  system and genes for carotenoid and bacteriochlorophyll synthesis, with a highly conserved synteny compared to the other Rhodopseudomonas genomes. Similar to other Rhodospseudomonas genomes, the HC1 genome also contains the transcriptional regulators PpsR1 and PpsR2 that repress photosystem development under high aeration conditions [Rhodopseudomonas had developed a unique and sophisticated set of photoreceptors and light responsive regulators to respond to changes in light quality and intensity [nditions . The genntensity ,15,16,48The synteny downstream from the photosynthetic gene cluster in strain HC1 is only conserved in strain ATH 2.1.18, which is also the closest relative based on the WGS comparison . Strain Rhodopseudomonas is unique amongst the purple nonsulfur bacteria in the fact that the reaction center-LH1 complex can both obtain an open and a closed confirmation [irmation . The opeRhodopseudomonas. Some have protW clustered with an NADH:flavin oxidoreductase (as in HC1) while others have an N-ethylmaleimide reductase  for carbon fixation: both genes encoding Form I Cbb large and small subunits, and Form II CbbM larger single RubisCO proteins are present in the genome. The presence of two forms of RubisCO is similar to other docyclus ,52. Five3 oxidase, cytochrome aa3 oxidase and a cytochrome bd ubiquinol oxidase are all present in strain HC1, similar to other Rhodopseudomonas strains. It also has cytochrome P450 genes for substrate hydroxylation and pyrroloquinoline quinone (PQQ)-dependent periplasmic dehydrogenases. Strain HC1 also has a complete set of nap, nir, nor for denitrification. The nos gene cluster (seven genes) that allows growth under anaerobic dark conditions with nitrous oxide as a terminal electron acceptor is missing from strain HC1. Most Rhodopseudomonas strains have the nos genes, except for strains AAP120, BisB5, B29 and faecalis PSBS. Strain HC1 also contains the genes for the low-oxygen sensor regulatory system FixLJ-K, which is present in all Rhodopseudomonas and widely distributed in the rhizobiaceae.Genes for cytochrome cbbRhodopseudomonas strain tested so far that was able to use L-arginine as a sole carbon source for heterotrophic growth  genes and one iron-iron nitrogenase  were annotated, besides several nif assembly and regulatory proteins. Nitrogenases convert nitrogen gas to ammonia and generate hydrogen gas as part of their catalytic mechanism. The presence of several nitrogenase subunit genes in strain HC1 indicates its ability for nitrogen fixation and hydrogen production, as expected for a Rhodopseudomonas species.As expected for Rhodopseudomonas has been recognized for its distinctive ability to degrade aromatic compounds [Rps. palustris type strain DSM 123 and Rps rutila type strain R1, and several other strains. The HC1 genome is indeed also lacking the cluster of genes for the benzoate degradation pathway (RPA0650–RPA0673), similar to the closely related strains AAP120 and 2.1.18, and HaA2.ompounds ,12. Howeompounds . We founRps. strain HC1 genome, identified a cluster of four genes related to aromatic PCB degradation that is only present in this strain. The gene cluster consists of a 4-hydroxyphenylacetate-3-monoxygenase (PGF_00701895) and a catechol-1,2-dioxygenase  preceded by two shorter genes encoding a flavin reductase and a putative ester cyclase. 4-hydroxyphenylacetate-3-monoxygenase has been shown to be important in aromatic PCB degradation and attacks a broad spectrum of phenolic compounds. It uses FADH2 as a substrate, produced by a flavin reductase [Bradyrhizobium sp. strain 41S5, which is a soybean nodulating bacteria found in eastern Canada [Bradyrhizobium genome.Interestingly, a closer comparison of unique genes in the eductase , which ieductase ,55,56. Tn Canada , althougThe presence of this unique gene cluster indicates that strain HC1 is capable of the degradation of some specific aromatic PCB-type chemicals with potentially interesting biotechnological applications ,56. FurtRhodopseudomonas strains are known to have natural resistance to some antibiotics [There is a growing global concern with increasing antibiotic resistance and consequently the search for antibiotic resistance signatures is also becoming important in environmental samples ,59,60. Aibiotics , requiriibiotics ,62. A geRhodopseudomonas infernalis sp. nov. for this novel species.Based on the genomic, genetic and physiological differences described above, we propose that strain HC1 belongs to a new species and propose the name Rhodopseudomonas infernalis .Rps. palustris [a (near IR maxima at 806 and 859). Extracted carotenoids are of the spirilloxanthin type. Photoheterotrophic growth in anaerobic conditions and chemothrophic growth under dark oxic conditions is possible with a variety of organic substrates: malate, tartrate gluconate, fumarate, aspartate, glutamate, casamino acids, arginine, and D-glucose . Cells appear to widen at the poles, creating a dumbbell shape . Cells -glucose . No growStrain HC1 was isolated from pond water at the Hell Creek watershed in Omaha, Nebraska, US.Rhodopseumonas based on genomic and physiological comparisons, for which we proposed the name Rhodopseudomonas infernalis sp. nov. The species showed both phototrophic and chemotrophic growth on a wide range of organic substrates and the genome also contains genes for the degradation of yet unidentified aromatic PCB-type chemicals. The genome also contains several nitrogenases, consistent with Rhodopseudomonas ability to fix nitrogen, which stimulates the growth of plants and microbes.A new photosynthetic strain was isolated from the Hell Creek native restoration area in suburban Omaha, NE. The isolate was found to be a new species of Rps. infernalis HC1 indicate that its presence at the Hell Creek restoration area contributes to N2 and carbon fixation and plant growth and plays a positive role in the degradation of anthropogenic substances. However, the genome also contains several antibiotic resistance genes, indicating a broad range of antibiotic resistance in this environmental isolate. The presence of antibiotic resistance genes in new isolates from urban native restoration areas contributes to a growing global problem of wide-spread environmental traces of antibiotic resistance.The physiological and genomic features of


Yersinia IgA were identified as potential predictive markers for slower treatment responses in CSU. In conclusion, the present study highlights the efficacy of omalizumab for the treatment of chronic urticaria. With emerging new therapeutic options for chronic urticaria, further genetic as well as molecular markers are needed to establish patient-specific therapy selection. Omalizumab, a monoclonal antibody targeting IgE, has been approved in 2014 for the treatment of H1 antihistamine-refractory chronic urticaria. Data on long-term effectiveness and predictive factors for treatment response are currently limited. In this monocentric, prospective, observational study, 112 patients with chronic spontaneous urticaria (CSU) and 32 patients with chronic inducible urticaria (CIndU) were included. In addition to a rapid response, omalizumab also showed good effectiveness on both forms of chronic urticaria over 2 years. Low total IgE and elevated  Chronic urticaria is manifested by wheals and/or angioedema that recur for at least 6 weeks. This occurs either spontaneously or as a result of external stimuli, e.g., cold or pressure. Accordingly, two subtypes are differentiated: chronic spontaneous urticaria (CSU) and chronic inducible urticaria (CIndU) . With reDue to the identified relevance of IgE and FcεRI, studies investigating the efficacy of omalizumab, a monoclonal anti-IgE antibody, followed. The phase 3 study showed a significant reduction of itch after 12 weeks compared to placebo . OmalizuTo date, prospective data from real-world care are limited. Ertas et al. demonstrated that initial serum total IgE can be used as a predictive marker for response . In the Helicobacter stool antigen test, Yersinia serology , differential blood count, CRP, TSH, thyroid autoantibodies , total IgE, and Sx1 . Furthermore, an autologous serum test was performed in a large proportion of patients [The present study is a monocentric, prospective, observational study. The study population was formed from all adult patients who completed the preliminary examinations and received at least one injection of omalizumab between January 2014 and January 2020 in the Department of Dermatology, Allergology and Venereology of the Hannover Medical School (MHH). Before starting therapy, a variety of clinical as well as laboratory parameters were collected and evaluated in the analyses. Clinical parameters were gender, smoking status, type of urticaria, age at onset, and age at start of therapy. Laboratory chemistry included patients . At each2-test. For comparison of changes from baseline, ANOVA was performed followed by Bonferroni-corrected post-hoc analysis. For consideration of factors influencing response, “responder” was defined as improvement in UCT score by at least 3 points at 1 month. This corresponds to the described minimal clinical difference (MCD) for UCT [General characteristics were analyzed using descriptive statistics and statistically compared according to scale level using the Mann-Whitney U test and the χ for UCT . Binary From 2014 to 2019, therapy with omalizumab was documented in 161 patients. 17 patients were excluded for further analysis because (1) the diagnosis included only histamine-mediated angioedema (n = 7), (2) initiation did not occur at MHH (n = 4), (3) the patient initially received omalizumab as part of a study (n = 4), and (4) the diagnosis changed during the course (n = 2). Of the 144 patients, 112 (77.8%) experienced CSU exclusively and in 53.6% with concomitant angioedema . In 22.2To analyze the effectiveness of omalizumab for the treatment of chronic urticaria, the change in UCT as well as CU-Q2oL after 1, 2, 3, 6, 12, and 24 months were considered first. There was a significant improvement in urticaria control as well as quality of life already after 1 month A. A furtWith regard to the CIndU patients, a significant improvement in urticaria control and quality of life was also observed after 1 month B. ComparOmalizumab therapy was well tolerated by all patients. No relevant adverse events were reported. To analyze the extent to which pre-treatment testing and clinical characteristics can predict a rapid response to omalizumab, the CSU study population was divided into responders and non-responders (at least 3 points improvement in UCT after 1 month) . This shYersinia IgA with improvement in quality of life at 2, 6, and 12 months was evident, i.e., increased Yersinia IgA was associated with reduced improvement in quality of life (as well as control of urticaria at 12 months). Furthermore, long-term response over 12 months correlated negatively with current age and age at initial manifestation. For further analyses regarding the influence of different factors on the improvement of urticaria control over time, a correlation matrix was constructed . In partThe results of this prospective long-term observational study underpin the effectiveness of omalizumab for the treatment of CSU as well as CIndU and confirmed the role of serum total IgE before therapy as a predictive factor for treatment response, especially after 1 month. Since the phase 3 trial of omalizumab and subsequent approval for the treatment of CSU inadequately responsive to H1 receptor blockers, there have been a few reports of treatment effectiveness under real-world conditions. Nettis et al.  describeYersinia IgA concentration, i.e., higher levels correlated with less improvement in CU-Q2oL at 6 months and UCT and CU-Q2oL at 12 months. Recently, an association of low IgA with low IgE in CSU was demonstrated and postulated [Surprisingly, with respect to response to omalizumab, we found a negative correlation with stulated . We coulThe present results confirm the generally very rapid response of CSU and CIndU to omalizumab already after the first dose, but also show that it is worthwhile to treat beyond 3 months because several patients benefit from this. Even over 2 years of treatment, effectiveness and improvement in quality of life are significantly improved and stable in CSU and CIndU . Our results showed female gender as a positive predictor of rapid response. In contrast, Fok et al.  found noIn terms of limitations, the small number of CIndU patients should be mentioned. In addition, the significance of CSU is also limited in individual subgroup analyses with low frequencies. Overall, however, this study provides insight into the effectiveness in clinical practice. Since 2019, self-application of omalizumab has been approved from the fourth dose onwards, so that the systematic assessment of long-term efficacy with standardized instruments has been more difficult in everyday clinical practice since then, because the patients are no longer under such close medical supervision. In conclusion, the present study highlights the effectiveness of omalizumab for the treatment of chronic urticaria – CSU as well as CIndU. In particular, total IgE has been identified as a predictive marker for response in CSU. Real-life evidence, as presented in this study, is of great importance to evaluate the effect of a drug in real-world use. Especially with the emerging new systemic therapies for CSU, comparative analyses to other drugs in such observational studies become even more important. None. S. Traidl declares no conflict of interest. B. Wedi received fees for lectures or advisory boards of several hours from ALK-Abéllo, Bencard, Biocryst, CSL Behring, HAL Allergy, Novartis, Takeda, ThermoFisher Scientific.