As sessile organisms, plants have evolved complex mechanisms to rapidly respond to ever-changing ambient temperatures. Temperature response in plants is modulated by a multilayer regulatory network, including transcriptional and post-transcriptional regulations. Alternative splicing (AS) is an essential post-transcriptional regulatory mechanism. Extensive studies have confirmed its key role in plant temperature response, from adjustment to diurnal and seasonal temperature changes to response to extreme temperatures, which has been well documented by previous reviews. As a key node in the temperature response regulatory network, AS can be modulated by various upstream regulations, such as chromatin modification, transcription rate, RNA binding proteins, RNA structure and RNA modifications. Meanwhile, a number of downstream mechanisms are affected by AS, such as nonsense-mediated mRNA decay (NMD) pathway, translation efficiency and production of different protein variants. In this review, we focus on the links between splicing regulation and other mechanisms in plant temperature response. Recent advances regarding how AS is regulated and the following consequences in gene functional modulation in plant temperature response will be discussed. Substantial evidence suggests that a multilayer regulatory network integrating AS in plant temperature response has been unveiled. Arabidopsis thaliana, 60% in Drosophila melanogaster and more than 95% in humans were reported to be alternatively spliced, greatly increasing the complexity of transcriptome and proteome .,47.′ splice site a .From a pre-mRNA′ perspective, the splicing process is simply accomplished by two transesterification reactions ,47. In te intron a [47]. Itogether a [47]. Tc center . The curc center ,51,52. Sice site b 53];  was significantly associated with AS pattern changes [Chromatin status  is usually considered as being related to transcriptional regulation. Furthermore, many recent studies in animal systems have shown that epigenetic mechanisms can directly affect gene splicing ,55,56. T changes .FLM, MAF2, and FCA, as well as circadian clock regulators PRR3 and PRR7 [It has long been known that temperature changes can lead to genome-wide epigenetic alterations ,73,74,75and PRR7 . Indeed,and PRR7 . By usinand PRR7 ,69.Co-transcriptional splicing provided a mechanistic basis for the cis-regulatory effect of chromatin status on AS, as it links splicing and chromatin spatially and temporally ,14,77,78trans factors, splicing factors were reported to promote or repress splicing by interacting with cis-elements (sequence motif such as splicing enhancer or splicing silencer) on pre-mRNA [cis-elements on pre-mRNA to promote constitutive splicing or mediate alternative splicing, while the RS domain mediates protein–protein interactions to activate splicing [Splicing factors (SFs) refer to RNA-binding proteins which associate with pre-mRNA and core spliceosome in constitutive splicing or alternative splicing ,22,83. Apre-mRNA ,20,43,83splicing . It has splicing . SR protsplicing ,87,88. Tsplicing .FLM, contributing to temperature-responsive flowering in Arabidopsis [sf1 mutants are hypersensitive to heat stress and displayed impaired splicing pattern of HSFA2 gene and altered expression of HSFA2 targets [CCA1 [CCA1 in response to high temperature, suggesting the potential role of SR45 in diurnal temperature-dependant splicing of CCA1 [Several studies have revealed the important roles of splicing factors in plant temperature response ,90. For bidopsis . In addi targets ,35,36. Hts [CCA1 . SR45 mu of CCA1 .PCP mutation caused striking development deficit, specifically under low temperature [FLM, in response to low temperature [Sta1 mutant plants showed defects in the splicing of temperature-responsive genes COR15A, HSFA3 and HSA32 [Spliceosome components and associated proteins were also involved in modulating AS in plant temperature response. The protein core of spliceosome is composed of Sm ring proteins and the related Sm-like complex ,93. Sm pperature . Additioperature . RBP45d,perature . STABILIperature ,99. Sta1nd HSA32 ,99.SR genes in tomato [SR genes were also reported to undergo AS in response to low temperature [Splicing factors and spliceosome components can be regulated by temperature changes via different mechanisms ,43,100. n tomato . A CLK kn tomato . Moreoveperature . Many SRperature . In addiperature .HSF-A2 was reported to give rise to different splicing isoforms depending on environmental temperature [HsfA2-II, which can be degraded through NMD [ELF3 (EARLY FLOWERING 3), which is part of the evening complex, leading to transcripts that are targeted by NMD [LHY (LATE ELONGATED HYPOCOTYL) gene, resulting in a nonfunctional LHY transcript that contains a PTC and is degraded by NMD [AS can generate transcript isoforms with different stability, localization or polyadenylation sites, and thus leads to different functionality ,105,106.perature . A heat-ough NMD . AS–NMD d by NMD . Cold std by NMD .SR30, RS2Z33 and the SEF factor, the intron-containing transcripts were found predominantly sequestered in the nucleus to escape the NMD pathway [In plants, intron retention is the most frequent AS event and usually leads to PTC-harboring transcripts, but only a subset of them can be degraded by the NMD pathway ,112,113. pathway ,117. Eme pathway ,118. In  pathway . These t pathway . Thus, iFLM (FLOWERING LOCUS M) gene is subject to temperature-dependent AS and produces two protein-coding splice variants, FLM-β and FLM-δ. These two AS transcripts can be translated into proteins with opposing functions, acting as a repressor (FLM-β) and promoter (FLM-δ) of flowering [FLM-β is dominantly expressed to form a protein complex with transcription factor SVP and represses flowering [FT and SOC1 genes from repression and thereby promoting flowering [A major effect of AS is that it dramatically increases the complexity of proteome by producing transcripts with different protein-coding potential . Indeed,lowering ,121,122.lowering . At elevlowering . A dominlowering ,123. In lowering ,110.HSF-A3B-III, which encoded a truncated protein interacting with full-length HSFA3 to reduce the adverse effects of excessive HSFA3 accumulation, enhancing tolerance to prolonged heat stress in lily [SGR5 gene produces two protein variants, full-length SGR5A dominant negative effect was also reported to modulate plant heat stress response. Prolonged heat stress in lily can induce the production of an AS isoform  in lily . In Arabed SGR5β . High tece stems .TRPV1 gene produces a channel with a truncated carboxy-terminal cytoplasmic domain, which is hypersensitive to infrared and can be activated at as low as 30 °C [HSFA2 AS isoforms, HSFA2-I and HSFA2-II, encode proteins with similar transcriptional activation activity under heat stress [HSP genes transcription [SR45 gene possesses two splicing isoforms, SR45.1 and SR45.2, which encode proteins differing by eight amino acids, including several putative phosphorylation sites [SR45.1 can rescue the floral but not the root phenotype in Arabidopsis sr45 mutant, while SR45.2 could complement the root growth defect [A dominant negative effect only offers more regulatory flexibility to fine-tune gene functionality. AS can also generate protein variants with distinct functions contributing to functional diversification . An inteas 30 °C . This spas 30 °C . Howevert stress . Howevercription . The Araon sites ,129. Remh defect .Extensive studies revealed that AS can couple to various mechanisms to modulate gene function, which dramatically extend the AS regulatory network in eukaryotes. For example, AS can be coupled to miRNA-related regulation by either modulating pre-miRNA splicing to control miRNA production or producing AS isoforms of miRNA targets with different sensitivity to miRNA binding ,131,132.TaVRN1 has revealed that lncRNA VAS is derived from the sense strand of TaVRN1 by AS during vernalization [VAS was physically associated with transcription factor TaRF2b to promote TaVRN1 expression and flowering [Plant temperature response is a systematic reaction involving various molecular mechanisms. It is reasonable to speculate the coupling of AS to different mechanisms in plant temperature response. However, very few such studies have been reported. Interestingly, a recent study of the plant vernalization gene lization . VAS waslowering .To date, extensive studies have reached a solid conclusion that alternative spicing is an essential component in plant temperature response , from reBy what mechanisms temperature signals are passed to splicing control remains unclear. It is well known that several transcription factors are master regulators in the transcriptional control of temperature response . For exaIntron retention is the most frequent AS event in plants and prevalently generates transcripts with PTC . Theoret


ACTC1, lincRNA-AMY2-STAT1, and/or lincRNA-AMY2/miR-204/STAT1 were sieved, and the results indicate that lncRNA could participate in IMF deposition through direct regulation or ceRNA. These findings provide a basis for analyzing the molecular mechanism of IMF deposition in pigs and lay a foundation for developing and utilizing high-quality resources of local pig breeds.Long non-coding RNA (lncRNA) participates in the regulation of various biological processes, but its function and characteristics in intramuscular fat (IMF) deposition in different breeds of pigs have not been fully understood. IMF content is one of the important factors affecting pork quality. In the present study, the differentially expressed lncRNAs (DE lncRNAs) and their target genes were screened by comparing Queshan Black (QS) and Large White (LW) pigs based on RNA-seq. The results displayed 55 DE lncRNAs between QS and LW, 29 upregulated and 26 downregulated, with 172 co-located target genes, and 6203 co-expressed target genes. The results of GO and KEGG analysis showed that the target genes of DE lncRNAs were involved in multiple pathways related to lipogenesis and lipid metabolism, such as the lipid biosynthetic process, protein phosphorylation, activation of MAPK activity, and the Jak-STAT signaling pathway. By constructing regulatory networks, lincRNA-ZFP42- Pork is a crucial part of people’s diet. With the improvement in quality of life and changing dietary preferences, people introduce higher requirements for pork meat quality, nutrient content, and taste. Intramuscular fat (IMF) is a vital trait that determines meat quality; its content is positively correlated with meat quality, and it is one of the key indexes to determine meat quality . MeanwhiPPARγ, thus promoting adipocyte differentiation [KLF6 knockdown may affect the formation and lipid metabolism of bovine adipose cells through various biological and molecular pathways such as calcium and cAMP signaling pathways [C/EBPα is a key transcriptional regulator of adipogenesis, and it promotes adipogenesis by affecting the H3K4me3 and H3K27me3 histone modifications of the C/EBPα locus and activating cis-C/EBPα transcription [PPARγ and C/EBPα, thus inhibiting adipogenesis, and the PU.1 antisense strand can transcribe the un-encodable transcript PU.1 AS lncRNA, which combines with PU.1 mRNA to form a complex mRNA/AS lncRNA, hinting at the translation of PU.1 and promoting adipogenesis [FNIP2 axis in chicken abdominal adipocytes [lncRNAs are RNA transcripts larger than 200 nucleotides in length and with no coding ability that were previously considered transcriptional noises without biological function . Howeverntiation . Studiespathways . C/EBPα cription . PU.1 ovogenesis ,13. LncIogenesis . By seleogenesis . After aogenesis . The anaogenesis . LncIMF2ogenesis . LncRNA-ipocytes . LncRNA ipocytes .Pigs are highly similar to humans in anatomy, physiology, and genome, making them ideal animal models for biomedical research . TherefoThe QS and LW pigs used in this experiment were obtained from Henan Fenghua Breeding Share Limited Company and were fed under the same feeding and management conditions. Three QS and three LW with a weight of 100 kg were selected and fasted for 24 h. After slaughtering, LD samples at the 6th to 7th ribs on the right were collected for RNA extraction and placed in liquid nitrogen immediately. Then, all samples were stored at −80 °C for further analysis.® spectrophotometer . RNA quality detection, RNA sequencing, quality control, and mapping have been described in detail in our previous publication [Total RNA was isolated using TRIzol reagent  according to the manufacturer’s instructions. RNA purity was checked using a NanoPhotometerlication . HISAT2 lication .Known transcripts and transcripts less than 200 bp in length were filtered out. We determined whether the encoding potential was the key condition to determine lncRNAs. CNCI (v.2), CPC2 (v.0.1), and Pfam-sca (v.1.3) were used to screen the transcripts, and transcripts predicted with coding potential by either/all of the three tools mentioned above were filtered out ,27,28. Tp < 0.05 and |log2 fold change| ≥ 1.StringTie (v.2.1.1) was used to calculate the fragments per kilobase of transcript per million mapped reads (FPKMs) of both lncRNAs and coding transcripts in each sample . The BalIn this study, the co-expression and co-localization genes of lncRNA were predicted to explore the functions of lncRNAs. We retrieved the genes 100 kb upstream and downstream of lncRNAs and analyzed their functions. “Trans-acting” refers to the co-expression relationship between lncRNA and mRNA. To explore the trans roles of these molecules, we used a custom script to calculate the Pearson’s coefficient between the lncRNAs and coding genes. The correlation coefficient was greater than 0.95 to predict lncRNA target genes.p-value of less than 0.05 were considered to be significantly enriched in differential expressed genes. KEGG is a database resource for understanding the advanced capabilities and utilities of biological systems [https://www.omicstudio.cn/tool (accessed on 19 November 2022), The pathway with |NES| > 1, NOM p-val < 0.05, FDR q-val < 0.25 was considered statistically significant.GO enrichment analysis of the related genes of DE lncRNAs was performed using KOBAS. GO terms with a corrected  systems . We perf systems . The res systems . Gene seDE lncRNAs and their co-expressed target mRNAs which may play a role in the regulation of intramuscular fat deposition were screened, and the interactions between miRNAs and DE lncRNAs and mRNAs were analyzed using miRanda software  to constThe differentially expressed genes were subjected to protein–protein interaction (PPI) analysis based on the STRING database, which is known and predicted for PPIs. The screened differential genes were imported into STRING for online analysis, and the corresponding data were imported into Cytoscape (v.3.9.1) for MCODE (v.2.0.0) analysis. The parameters were set as degree cutoff = 2, node score cutoff = 0.2, k core = 2, maximum depth = 100, and the score was set to ≥4 to obtain the two closest clusters from the PPI.® Premix Ex Taq™ II  on the CFX96 Real-Time PCR Detection System . Glyceraldehyde3-phosphate dehydrogenase (GAPDH) was used as the normalization control, and all reactions were carried out in triplicate. The 2−ΔΔCT method was used to quantify relative expression levels.We randomly selected five lncRNAs and five mRNAs to validate their expression. Approximately 1 μg of each RNA sample was used in the PrimeScript™ RT reagent kit with gDNA Eraser  to convert the total RNA to cDNA. Next, qRT-PCR was performed using TB GreenIn this study, we obtained the raw reads by RNA-seq and filtered out articulated, null, and low-quality sequences. Clean reads obtained in each library accounted for 94.33%–95.20% of the raw reads [Of the 55 DE lncRNAs detected, 29 were upregulated, and 26 were downregulated A. A totaIn order to understand the potential functions of novel lncRNAs, we conducted co-localization and co-expression analysis of candidate DE lncRNAs. Among the 55 DE lncRNAs, the co-localization analysis revealed 45 DE lncRNAs corresponding to 172 related mRNAs, and the co-expression analysis revealed 55 DE lncRNAs corresponding to 6203 related mRNAs. Further analysis showed that 7 DE lncRNAs were co-located with 8 DE mRNAs, and 55 DE lncRNAs were co-expressed with 326 DE mRNAs. It was obtained by enrichment analysis that the DE target genes co-localized with DE lncRNAs were mainly involved in muscle contraction regulation, cell differentiation, thyroid hormone synthesis, and cellular response to the lipopolysaccharide and p53 signaling pathway A. The DEGO analysis of co-localized DE target genes showed that, skeletal muscle contraction, apolipoprotein binding, calcium channel regulator activity, skeletal muscle cell differentiation, regulation of I-kappaB kinase/NF-kappaB signaling, gluconeogenesis, muscle contraction, and other items were significantly enriched . The resGO enrichment analysis of co-expressed DE target genes showed that the main enrichments were positive regulation of gene expression, muscle contraction, cellular response to lipopolysaccharide, skeletal muscle contraction, regulation of myoblast differentiation, lipid biosynthetic process, regulation of cell population proliferation, phosphatidylinositol phosphate binding, and other terms . The resGene Set Enrichment Analysis (GSEA) of DE lncRNAs co-expressed target genes showed that the insulin signaling pathway, FoxO signaling pathway, and Jak-STAT signaling pathway were also highly enriched A–C, whicLZTR1, CFL1, AGRN, STAT6, ZNF148, STAT1, LMNA, and so on. These DE lncRNAs and mRNAs may be potential key genes for the regulation of IMF deposition. Each lncRNA can be associated with one or more mRNAs, such as LNC_000040-ERCC2, LNC_003934-ACTC1, AGRN, and LMNA. Each mRNA can also be associated with one or more lncRNAs, such as MAPK1-LNC_009261, ACTC1-LNC_002449, LNC_000314, and LNC_003934.The lncRNA-mRNA co-expression network consisted of 126 network nodes with 427 junctions between 55 DE lncRNAs and 71 DE mRNAs A. The ceSTAT1, STAT6, AGRN, LMNA, and PIK3CD were the predicted target genes of miRNA. One lncRNA could bind to one or more miRNAs, and each miRNA could bind to one or more lncRNAs, where LNC_006333 adsorbed both ssc-miR-149 and ssc-miR-204, constituting the LNC_006333/ssc-miR-149/STAT6 and LNC_006333/ssc-miR-204/STAT1 regulatory networks.The constructed DE lncRNA-miRNA-mRNA ceRNA regulatory network included 6 DE lncRNAs, 8 DE miRNAs, and 39 DE mRNAs, as shown in STAT1, STAT6, IL6, and TBK1, where IL6 is the core gene. Module 2 consists of ACTC1, PIK3CD, TLN1, and CAPN3, where ACTC1 is the core gene  gene, to regulate porcine precursor adipocyte differentiation [FAAH) gene transcriptional activity in a trans-regulatory manner, while MSTRG.25116.1 was found to also act as a molecular sponge that competitively binds to gga-miR-1635 to promote the post-transcriptional expression of FAAH and participates in the regulation of chicken fat formation [SYISL, a conserved lncRNA in pigs and humans, can act as a sponge for miR-23a-3p, miR-103-3p, and miR-205-5p to increase the expression of the muscle atrophy inducer genes FoxO3a, MuRF1 and Atrogin-1, thus participating in the cross-species muscle atrophy process [In this study, the expression level of lncRNAs in the LD of QS and LW pigs was analyzed by RNA-seq, and 55 DE lncRNAs were identified. lncRNAs have no coding ability and can play a role by binding to proteins or chromosomes, especially transcription factors . Huang e buffalo . It was ransport . lncRNA ntiation . By cons atrophy . In addiy [SIRT1 . FDNCR1 ntiation . MSTRG.2ormation . SYISL,  process . TherefoMAPK1, IL6, and PIK3CD are involved in the significantly enriched terms above and are associated with lipid metabolism, muscle development, inflammatory response, and cell proliferation migration, respectively [In order to further explore the potential functions of DE lncRNAs in pork quality regulation such as IMF deposition and muscle growth and development, we analyzed the intersection of DE mRNAs and target genes of DE lncRNAs by GO and KEGG enrichment analysis. GO results showed that DE lncRNAs are involved in muscle contraction, response to muscle activity, skeletal muscle cell differentiation, sarcomere organization, and myofibril development. Muscle consists of muscle fibers, which serve as the material basis for meat quality traits, and its type is closely related to meat quality, which is an important parameter for the evaluation of meat quality ,45. Studectively ,52,53. Tectively . ECM–recectively . GSEA reectively ,57,58.STAT1, STAT6, IL6, and TBK1. The STAT family plays a regulatory role in adipogenesis [STAT1 is related to adipocyte differentiation and affects the process of lipid metabolism [STAT1 and binds to miR-204, and STAT1 is the target gene of miR-204 and participates in many pathways related to lipid metabolism. Therefore, LNC_006333 can directly regulate gene expression and exert its biological function through ceRNA, thus affecting the expression level of STAT6, IL6, and TBK1 while regulating STAT1. LNC_006333 is a lincRNA between PRMT6 and AMY2, and AMY2 is the nearest protein-coding gene to LNC_006333. Therefore, it is named lincRNA-AMY2, and lincRNA-AMY2-STAT1 and/or lincRNA-AMY2/miR-204/STAT1 may be an important regulatory network that affects fat deposition in pigs. In the present study, lncRNAs such as EAD1-AS (LNC_004524), lincRNA-STRIT1 (LNC_002449), HNRNPH3-IT (LNC_003082), and IBA57-IT (LNC_004931) regulate fat deposition in the above manner. ACTC1 is involved in the regulation of the proliferation cycle of bovine myogenic cells, which is positively correlated with myogenesis and promoting adipocyte differentiation, thus playing a biological function in muscle development and fat deposition [SLC7A2 and FAT1, is a lincRNA, named lincRNA-FAT1 because it is the closest to FAT1. It directly regulates ACTC1 and affects the expression levels of PIK3CD, TLN1, and CAPN3. In addition, lncRNAs such as PLP1-IT (LNC_009595), lincRNA-PPP2R5A (LNC_009318), lincRNA-RORA (LNC_000187), and lincRNA-MIR99A (LNC_002605) regulate the fat deposition process in the same way. In summary, the regulation of different lncRNAs differs. Through a series of analyses, this study screened regulatory networks such as lincRNA-AMY2-STAT1 and/or lincRNA-AMY2/miR-204/STAT1, and lincRNA-FAT1-ACTC1 may affect IMF deposition by regulating adipocyte proliferation and differentiation and, thus, regulate pork quality. The results of this study provide an important reference for uncovering the regulatory mechanisms of intramuscular fat deposition in pigs and lay the foundation for further understanding the genetic mechanisms of pork quality formation.In the present study, the construction of the PPI interaction network revealed a high degree of correlation and a strong linkage between four genes, namely, ogenesis ,60. As atabolism ,62, and tabolism . LNC_006position . LNC_003ACTC1, lincRNA-AMY2-STAT1, and/or lincRNA-AMY2/miR-204/STAT1 were filtered out, which were potentially related to lipid deposition. These results provide a reference basis for analyzing the mechanism of intramuscular fat deposition in pigs.In the present research, based on the RNA-seq of LD muscle tissue of QS and LW pigs, 55 DE lncRNAs were identified, 29 upregulated and 26 downregulated. Through targeted prediction, we discovered that 172 target genes were co-located, and 6203 target genes were co-expressed. Functional enrichment analysis revealed that it was involved in many pathways related to lipid metabolism, such as the activation of MAPK activity, the lipid biosynthetic process, the thyroid hormone signaling pathway, and the Jak-STAT signaling pathway. The regulatory networks of lincRNA-ZFP42-


We also report a unique histopathology and mutational profile that may have important implications for the characterization and prognosis of SMZL.Standard treatment regimens for the management of patients with refractory splenic marginal zone lymphoma (SMZL) are currently unavailable. Here, we report a case of SMZL, which, after failing multiple therapeutics, demonstrated an impressive clinical response to combined Venetoclax and Velcade (V Splenic marginal zone lymphoma (SMZL) is a rare subtype of non-Hodgkin’s lymphoma that is thought to arise from memory B lymphocytes in the marginal zone of lymphoid follicles in the spleen . AlthougA 71-year-old African American male Vietnam Veteran presented with abdominal pain and dizziness in March of 2001. Computed tomographic scans (CT) showed hepatosplenomegaly with intra-abdominal LAD adjacent to the duodenum. Liver biopsy showed nodular lymphoid infiltrates composed of a mixture of small cells and scattered larger cells positive for CD20, kappa, BCL-2, IgD , and BCL6 (mostly on few larger cells); and negative for lambda, CD5, CD10, and MUM1. The Ki67 proliferative rate was up to 15% and a few larger lymphoid cells were positive for P53. Peripheral blood-flow cytometry demonstrated surface kappa-restricted monoclonal B cells positive for CD20, CD19, CD22, and CD79b and negative for CD5, CD10, CD23, CD11c, CD25, and CD103 . The oveThe patient was lost for a follow-up until May of 2002, when he presented with post-prandial abdominal pain and 5–10 pounds of weight loss. Complete blood-count (CBC) revealed a white-blood-cell count (WBC) of 9.8 k/cmm , hemoglobin (Hgb) of 12.6 g/dL , platelet count (Plt) of 180,000 k/cmm (152–375 k/cmm), and lactate dehydrogenase (LDH) of 294 U/L . Human immunodeficiency virus, hepatitis B, and hepatitis C were negative. Bone marrow (BM) biopsy showed hypercellularity for age, mainly due to erythroid and myeloid hyperplasia without left shift and small lymphoid aggregates (representing <10% of cellularity) with interspersed large lymphocytes, morphologically and immunophenotypically congruent lymphomatous involvement. CT scan revealed splenomegaly measuring 14 cm with an enlarged para-aortic lymph node (LN) (largest diameter of 4 cm). In September of 2002, after 4 weekly doses of rituximab monotherapy, CT monitoring revealed normalization of spleen size and some residual (sub-centimeter) retroperitoneal LAD. Notably, the Lugano classification and response assessment criteria were used as recommended by Cheson et al. [As outlined in In December of 2011, a repeat BM biopsy showed increased lymphomatous burden (~50% of cellularity) without evidence of transformation. Karyotype analysis detected a new del (2) (q13q33) in addition to the complex karyotype previously observed: 45, XY, t  , del (2) (q13q33), add (4) (p14), del (5) (q13q15) [The patient received several courses of chemotherapy, including Rituximab monotherapy , splenectomy (2006), R-CVP (6 cycles in 2008), bendamustine and rituximab (2015), obinutuzumab and lenalidomide (2015–2016), ibrutinib alone initially, and then ibrutinib and rituximab (2016–2018). Note that some rituximab monotherapy courses were given per patient request at the time of disease progression.In April of 2018, a CT scan showed stable hepatomegaly with worsening of LAD (largest 8 cm) in the mediastinum, abdomen, and pelvis. Positron emission tomography (PET) showed avid FDG uptake  in abdominal LNs and several areas of focal uptake in the liver, initially concerning for transformation into an aggressive lymphoma. However, a liver biopsy demonstrated known MZL with a mildly increased Ki67 proliferative index without evidence of transformation to diffuse large B-cell lymphoma (DLBCL). One course of Idelalisib therapy was given in July of 2018, with a follow-up CT demonstrating an initial 25% reduction in abdominal LAD (10/18). However, this regimen was discontinued due to the formation of a colonic abscess requiring colectomy.In June of 2019, the patient complained of worsening abdominal pain and night sweats, and a CT scan revealed substantial hepatomegaly measuring 23 cm in greatest cranio-caudad dimension with stable LAD. A liver biopsy showed MZL again, with increased large cells and variable Ki67 proliferative rates up to ~20% in some areas without evidence of transformation. The patient was started on Venetoclax 50 mg PO in June of 2019, which was escalated to 400 mg PO without evidence of tumor lysis syndrome prior to adding weekly Velcade (V2). In October of 2019, a CT scan demonstrated a decrease in the size of intrathoracic, mesenteric, retroperitoneal, and deep pelvic LNs by 75–90%, as well as normalization of liver size when compared to prior studies. The patient reported subjective improvement in appetite, stabilization of weight, and no constitutional symptoms over subsequent follow-up visits. However, LDH values, which had been within normal limits, increased to 312 U/L in December of 2019. PET scan (01/20) demonstrated progression of previously identified lesions (increase by >50%) and numerous new lesions above and below the diaphragm, suggesting progression to a more aggressive lymphoma. A right axillary LN excision revealed known MZL with increased large cells without sheeting and a Ki67 proliferative rate focally increased to 30%. CD30 was negative and CD21/CD23 immunohistochemistry staining highlighted residual follicular dendritic meshworks ruling out transformation to DLBCL. Next-generation sequencing (NGS) of nodal tissue revealed the following mutations: CD36 I399fs*19, MYD88 L265P and V217F, MLL2 splice site 4237-57_4377del198, NOTCH2 R2400*, PHF6 M1K, TAF1 R843W , TRAF3 rearrangement exon 10, and several variants of uncertain significance  splice site 52 + 2T > C, CEBPA R327W, CIC P660L, CREBBP F1484C, DNMT3A F751I, FLYWCH1 rearrangement, GNA13 Q27H, HIST1H1E L62V, HIST1H2BC E72K, MKI67 Q2084P, NTRK1 V211M, PCLO T2697A, PDCD1LG2 (PD-L2) C248fs*35).2.At that time, treatment was discontinued. The patient died from SMZL in January of 2020 after an impressive clinical response for about 6 months with combination VHerein, a complicated case of SMZL in a 71-year-old African American male requiring multiple therapies during a 20-year prolonged course is presented. The patient’s clinical course was initially well controlled with multiple courses of rituximab monotherapy pre- and post-splenectomy. However, treatment resistance arose requiring additional intervention. Treatment with many therapeutics available for MZL achieved variable responses prior to disease progression to a more aggressive SMZL without transformation to DLBCL.SMZL is a rare disorder, affecting 1.8 per 1,000,000 person years, and the indolent nature of SMZL has undercut efforts to establish a standard treatment regimen. Asymptomatic patients with SMZL can be under observation without treatment for many years with no change in overall outcomes ,6. BasedRelapsed/refractory (R/R) patients with SMZL include those who failed local treatment (splenectomy) and those with disease progression following rituximab monotherapy. Retreatment of (R/R) patients with rituximab monotherapy is commonly employed as it retains its efficacy in most cases, however, consideration of rituximab-based chemotherapy regimens is also appropriate ,17,18,19Over the past 10 years, improvements in our ability to molecularly and genetically characterize MZL as it relates to underlying disease biology has led to the use of more targeted therapies with improved efficacy and tolerability. To this end, a few prospective studies investigating the use of targeted therapies for the treatment of MZL, including a small subset of SMZL, have shown promise.Small molecule kinase inhibitors targeting BCR signaling, such as the Bruton’s tyrosine kinase (BTK) inhibitor (ibrutinib), seem to provide new avenues of therapeutic strategies ,22,23,24The proteasome inhibitor bortezomib also has significant single-agent activity in R/R MZL with an ORR of 48% (CR 31%). Similar impressive results were noted when bortezomib was combined with rituximab in R/R MZL . An ORR 2 with resolution of hepatomegaly and approximately three months of symptom-free survival, providing anecdotal support for this combination therapy in refractory SMZL or a subset with increased large cells.Velcade and venetoclax is a promising combination therapy for the management of refractory multiple myeloma . VenetocAlthough some of the initial biopsies were limited in size, the histopathology (including the splenectomy specimen) was not classical for MZL, and best fits the so-called MZL with increased large cells , which h2 therapy may represent an effective therapeutic option for refractory SMZL in the absence of clinical trials to guide treatment approaches.In summary, we report upon an impressive clinical response in a patient with refractory SMZL to combination venetoclax and velcade, which is currently being studied in the context of refractory multiple myeloma. This observation suggests that V2), which is currently being studied in the context of refractory multiple myeloma. This observation suggests that V2 therapy may represent an effective therapeutic option for refractory SMZL in the absence of clinical trials to guide treatment approaches.The rare and indolent nature of SMZL has hampered efforts to discover effective treatment regimens, especially in patients with R/R disease. Clinical studies aimed at assessing the efficacy of various therapeutic combinations for the treatment of SMZL have proven useful; however, heterogeneity among treatment courses and limited sample sizes have limited our insight. Consequently, the discovery of novel treatment combinations for SMZL remains an area of active interest. We report upon an impressive clinical response in a patient with refractory SMZL to combination venetoclax and velcade (V


Listeria monocytogenes and Staphylococcus aureus and alsoGram-negative bacteria of Escherichia coli(O157: H7), E. coli(ATCC 25922), Salmonella enteritidis, and Salmonella typhimurium. Moreover,it was found that FeNPs exhibited superior peroxidase-like activityto catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine(TMB) to produce a blue color product, oxidized TMB (oxTMB), in thepresence of H2O2. The colorimetric AA detectioncould be carried out by making the solution color lighter owing tothe antioxidant property of AA. The quantitative detection of AA couldbe performed simply, selectively, and sensitively with FeNPs witha detection limit (LOD) of 0.5462 μM in a linear range of 30–200μM.It has been well known that metallic nanoparticles withstrikingproperties possess wide application prospects in the processes ofcolorimetric detection, catalysis, disease diagnosis and treatment,energy, wastewater treatment, remediation, and antibacterial activityin recent years. Herein, iron-based nanoparticles (FeNPs), metallicnanoparticles, were synthesized via a facile chemical reduction methodusing a hyperaccumulator plant. Also, their use in antibacterial activityapplications and colorimetric ascorbic acid (AA) detection was investigated.It was observed that FeNPs presented high antibacterial potency againstGram-positive bacteria of  With this developed method, it canbe possible to synthesize nanoparticles in a short time with highefficiency, which is one of the advantages of chemical methods, andalso the process cost and the formation of toxic byproducts can bereduced using plants, which is one of the advantages of biologicalmethods. In the literature, the metallic nanoparticles synthesizedby various methods could be effectively used in various applicationareas such as bioremediation and decontamination applications,1 biomedical applications,2 drug delivery,3 removal of various pollutants,4 catalyst applications,5 sensing applications,6 antibacterialactivity applications,7 battery applications.8 Among the metallic nanoparticles, iron-basednanoparticles have attracted much attention due to their outstandingproperties such as high reactivity, adsorption capacity, biocompatibility,and mechanical, chemical, and thermal stability.9 Additionally, they possess magnetic features, providingeasy separation from the environment via an external magnet.10 Because of the as-mentioned superior propertiesof iron-based nanoparticles, they have been widely utilized in theapplications of catalysis, antimicrobial, anticancer, biosensor, energy,and wastewater treatment.11 To the bestof our knowledge, it is the first time to evaluate iron-based nanoparticles,synthesized using a hyperaccumulator plant as a metal ion source,in antibacterial activity applications and colorimetric AA detectionin this study.Nanoparticles can be synthesizedby physical, chemical, and biologicalmethods or hybrid methods, which are combinations of several of them.In this study, a new nanoparticle synthesis method was developed thatcombines the advantages of chemical and biological methods. Accordingto this method, the iron-based nanoparticle (FeNP) synthesis can becarried out by the chemical reduction of the metal ions in the leachsolution prepared from the iron hyperaccumulator plant of 12 So, one ofthe most featured practical applications of iron-containing nanoparticles,metallic nanoparticles, is their utilization as antibacterial agentsin the literature.15 In this study, the antibacterial activity of FeNPssynthesized from a hyperaccumulator plant was investigated againstvarious food-borne bacteria.The researchers have focused on developing moreeffective antibacterialnanoparticles without side effects that are easy to enforce sincethe risk of biological and bacterial assaults, especially in the sectorsof food, food packaging, and water, has increased gradually in recentyears. The dimensions of metallic nanoparticles are between bulk materialsand molecules/atoms/ions, which interact with cells to make stableentities with less energy.16 Therefore, it is crucial todetect AA with a simple, convenient, inexpensive, and sensitive method.Various methods have been established and applied to detect AA, includingtitration, enzymatic methods, electrochemical techniques, fluorescenceand chemiluminescence methods, capillary electrophoresis, and chromatographymethods.18 The detection of AA has been able to applythese methods with good sensitivities; however, these methods havesome disadvantages such as the need for trained technicians, the requirementof expensive equipment or chemicals, and impractical and time-consumingoperations. Among the traditional methods, the colorimetric methodbased on a chromogenic substrate producing color upon oxidation inthe presence of the natural enzymes is more ideal and effective indetecting AA. However, natural enzymes are too sensitive to extremeexperimental conditions, and so they can lose their catalytic activityeasily at strong acidic/basic pHs and high-temperature values. Inaddition, there are some drawbacks of natural enzymes such as highcost, low stability, and difficulty in storage.19 Nanozymes are inorganic nanomaterials with a more effective enzyme-likecatalytic activity in comparison to natural enzymes because of theirhigh catalytic activity, low cost, high stability, and wide rangeof applications.20 Hence, to overcome thesedrawbacks, various nanozymes such as palygorskite@Co3O4 nanocomposites,21 polyacrylonitrile–CuOnanoflowers,22 platinum nanoclusters,23 gold nanoparticles,24 cobalt-doped carbon quantum dots,25 Cu–Agbimetallic nanoparticles,26 and Fe3O4/nitrogen-doped carbon hybrid nanofibers27 have been developed as peroxidase-like catalyststo catalyze the oxidation of TMB in the presence of H2O2 for the colorimetric AA detection in the past decade. Sincesome difficulties have been faced with the sensitive detection ofAA in complex biological media, the researchers are still focusingon improving the catalytic activity, sensitivity, selectivity, andstability of enzyme-mimetic nanomaterials. In this study, FeNPs synthesizedfrom a hyperaccumulator plant were used instead of a natural enzymein the oxidation reaction of TMB in the presence of H2O2, and the addition of the antioxidant agent AA to this reactionmedia enables simple, sensitive, and selective colorimetric detectionof AA.Ascorbic acid (AA) is a naturalwater-soluble vitamin, which isa puissant reducing and antioxidant agent that has important rolesin battling bacterial infections, detoxifying reactions, and the formationof collagen in fibrous tissue, teeth, bones, connective tissue, skin,and capillaries. AA concentrations in the human bloodstream are generallybetween 0.6 and 2.0 mg/dL; however, it varies from tissue to tissue.The deficiency of ascorbic acid (<0.4 mg/dL) in the body can causethe emergence of various diseases such as immunity decrease, anemia,and even scurvy. Taking more than the recommended amount of AA, whichis more than 2.0 g per day, can result in side effects such as nauseaand vomiting, diarrhea, abdominal pain and cramps, heartburn, insomnia,headache, fatigue, and kidney stones. Besides, AA has been graduallyutilized in industries due to its strong reducing and antioxidantcapacity.22.14, as a reductantagent, was added to the leach solution prepared from P. brutia instead of the synthetic iron salt solutionunder the required experimental conditions. The details of the synthesismethod and the results of some characterization studies were presentedin our previous work.28 In this study,additional characterization studies using a Zeta-sizer via the dynamiclight scattering technique (DLS), Fourier transform infrared (FTIR)spectra, and a vibrating sample magnetometer (VSM) were used to definethe synthesized FeNPs.According to the iron-based nanoparticle (FeNP)synthesis method developed by our team, NaBH2.2Salmonella enteritidis, Listeria monocytogenes, Escherichia coli O157:H7, E. coli (ATCC 25922), Staphylococcusaureus, andSalmonellatyphimurium bacteria species, which are widely usedand food-borne pathogens, were selected to determine the antibacterialactivities of FeNPs. In the scope of the culture and inoculum preparation,the bacterial cultures were grown on tryptic soy agar (TSA) slantsand kept at 4 °C. Isolated colonies obtained from the TSA slantswere inoculated into a tryptic soy broth (TSB) medium. The broth culturewas incubated at 37 °C for 24 h. The optical density of the culturewas adjusted between 0.08 to 0.1 at 625 nm to obtain an inoculum sizeof 1 × 107 colony-forming unit (CFU)/mL. The antibacterialactivity of FeNPs was investigated using the agar plate method. Thesurface of the agar plate (Mueller Hinton Agar) was inoculated byspreading the test microorganism over the entire surface. Then, ahole with a diameter of 6 mm was punched aseptically, and 0.0015 gof the test compound (FeNPs) and 30 μL of sterile distilledwater were introduced into the well. The Petri dishes were incubatedat 37 °C for 24 h. The test compound diffused into the agar andinhibited the growth of the test microorganism. The diameters of inhibitiongrowth zones were measured with a digital caliper.292.32O2 solution, and 100 μL of a 1.0 g/L FeNPsolution were added sequentially onto 250 μL of a 0.5 mM 3,3′,5,5′-tetramethylbenzidine(TMB) solution in an UV–vis spectrophotometer cuvette. Thissolution was named “control” by us. After that, thespectrum scanning of the “control” containing the oxidationproducts formed as a result of the oxidation of TMB was performedwith a UV–vis spectrophotometer at a 300–1100 nm wavelengthrange. Then, a series of control solutions were prepared, and 1000μL of AA solutions at different concentrations (1.0–500μM) were added separately to each prepared control solution.The reductions in the absorption peak intensities were determinedwith the addition of AA by performing repeat spectrum scans of theprepared solutions in the wavelength range of 300–1100 nm.For the determination of the minimum limit of detection (LOD) of FeNPs,the absorbance values of the control solutions containing the oxidationproducts (Ai) and AA solutions at differentconcentrations were added (Af) were recordedwith a UV–vis spectrophotometer at a 652 nm wavelength. Inorder to find the LOD value, a calibration line was formed by plottingthe different concentrations of AA against the absorbance changes(ΔA = Ai—Af) at these concentrations. The limit of detection(LOD = 3σ/s) was calculated according to the signal, which isequivalent to 3 times the standard deviation of the blanks, wheres is the slope of the calibration line and σ is the standarddeviation of the control solution (the solution containing the oxidationproducts without adding AA).30The antioxidant property of AA was utilized for thecolorimetric detection of AA. For this purpose, 500 μL of anacetate buffer solution (pH = 2.0), 500 μL of a 0.1 mM H2.42, CaCl2, ZnCl2, MgCl2, Al(NO3)3, (NH4)2HPO4, glucose, lactose, maltose, fructose,sucrose, urea, uric acid, ascorbic acid, oxalic acid, lactic acid, l-cysteine, glutathione, dopamine, and melamine. To determinethe selectivity of FeNPs, 500 μL of an acetate buffer solution(pH = 2.0), 500 μL of a 0.1 mM H2O2 solution,and 100 μL of a 1.0 g/L FeNP solution were added sequentiallyinto 250 μL of a 0.5 mM TMB solution, the solution containingall of the components was defined as “control”, in aUV–vis spectrophotometer cuvette. The absorbance values ofthe control solutions containing the oxidation products were recordedwith a UV–vis spectrophotometer at a 652 nm wavelength. Afterthat, the solutions of NaCl, KCl, CuCl2, CaCl2, ZnCl2, MgCl2, Al(NO3)3, (NH4)2HPO4, glucose, lactose,maltose, fructose, sucrose, urea, uric acid, ascorbic acid, oxalicacid, lactic acid, l-cysteine, glutathione, dopamine, andmelamine were added to each prepared control solution as a mixturewith AA (500 μL of 1.0 mM AA + 500 μL of 1.0 mM interferents).The absorbance values of the final solutions containing differentinterferents were recorded with a UV–vis spectrophotometerat a 652 nm wavelength, and the absorbance changes (ΔA = Ai– Af) were calculated to determine the selectivity of FeNPsand the interactions of different components.30The colorimetric detection of AA with FeNPs was carried out in thepresence of different components that can be found in real samplessuch as NaCl, KCl, CuClAi is theabsorbance change in the presence of the interferent i and Ar is the absorbance change in the presence ofreference that is distilled water.The recovery values were calculated with 2.5A = Ai– Af) werecalculated. The concentration values of AA were calculated from theobtained calibration line equation. Accordingly, the accuracy analysisof the method was performed by comparing the theoretical and calculatedconcentration values.30 The formulae for the recoveryvalue and the relative standard deviation (RSD) used in this analysisare given in In order to testthe applicability of the colorimetric detection of AA with FeNPs inreal samples, commercial AA-containing materials (vitamin C tabletsand vitamin C water) and AA solutions with known theoretical concentrationswere used. For this purpose, the “control solution”was prepared, and the absorbance values of the control solution containingthe oxidation products were recorded with a UV–vis spectrophotometerat a 652 nm wavelength. After that, a series of control solutionswere prepared, and 1000 μL of the solutions  was added to the control solutions containing theoxidation products. The absorbance values of the final solutions containingAA were recorded with a UV–vis spectrophotometer at a 652 nmwavelength, and the absorbance changes , remanent magnetization(Mr), and coercivity (Hc) values were obtained to be 4.5 emu/g, 0.8438 emu/g,and 125 Oe, respectively. The remanent magnetization value (Mr < 1.125) obtained for FeNPs, less than25% of the Ms value, showed that FeNPscould be easily separated from the media with a permanent magnet andcould be quickly dissolved in the solution without agglomeration.Since the Hc value of FeNPs synthesizedin this study was 125 Oe, it was determined that FeNPs were classifiedas semihard magnetic materials. It was concluded that FeNPs, whichwere classified as semihard magnetic materials, required more energythan soft materials to move in the loop and required a lower magneticfield than hard magnetic materials to reach saturation magnetization.Semihard magnetic materials have a wide range of uses such as magneticallycoupled devices , bias elements in productprotection/safety systems, relay magnets, magnetic tool holders, sensormagnets, magnetic stirrers, and level sensors.37 Accordingly,it can be said that FeNPs synthesized in this study could be usedin the application of semihard magnetic materials.A vibrating sample magnetometer (VSM) was usedto determine themagnetic properties of FeNPs by measuring the magnetization versusthe applied magnetic field (M–H) curve at room temperaturein the magnetic field range of ± 20 kOe. The hysteresis loopsshown in Ms) of FeNPs was compared with the iron-containing nanoparticlesinthe literature, and the results are summarized in Ms value than iron-containing nanoparticles in the literature. It wasthought that other components except for iron in FeNPs obtained inEDX analysis reduced the saturation magnetization (Ms) value of FeNPs.The saturation magnetization value , S. aureus, and S.typhimurium were determined as 20.85 ± 2.98,17.33 ± 2.62, 16.93 ± 1.80, 22.74 ± 1.09, 31.56 ±1.50, and 20.35 ± 1.46 mm, respectively. Accordingly, it wasproved that FeNPs were effective against all selected bacteria. Asa result, it was concluded that the strong inhibitory effect of FeNPssynthesized in this study against the tested food-borne microorganismswill allow FeNPs to be used as a preservative agent in the food industryto reduce food contamination and provide longer-lasting storage. Inthis case, it can be recommended to use FeNPs synthesized from thehyperaccumulator plant of P. brutia for bacterial-resistant coating and antibacterial applications forbiomedical devices.Theimages showing the antibacterial activity of FeNPs on test microorganismsand the graph showing the inhibition zone diameters obtained for eachmicroorganism are presented in The inhibition zone diameters of FeNPs were comparedwith the iron-containingnanoparticles in the literature, and the results are summarized in 3.32O2 via a Fenton-like reaction to generate •OH radicals,which could oxidize the chromogenic substrate TMB to the oxidizedTMB (oxTMB). The colorimetric detection of H2O2 with FeNPs as a peroxidase-like catalyst could be done by the spectrophotometricanalysis of the colored oxidation products formed at the end of thisreaction. For the detection of AA with FeNPs, AA could discolor theblue color of oxTMB to colorless-TMB by means of the antioxidant propertyof AA. The UV spectrum scans of the solutions given in According to the predicted colorimetric AA detectionsteps given in 2O2, a calibration line given in A = 0.001439 × CAA (μM) + 0.1973(R2 = 0.9894) with the AA concentrationin a range of 30–200 μM. The limit of detection (LOD)of FeNPs for the colorimetric AA detection was calculated to be 0.5462μM at an S/N  ratio of 3.0.In order to determine the LOD value of FeNPs forthe colorimetricdetection of AA via TMB oxidation in the presence of H3.42, CaCl2, ZnCl2, MgCl2, Al(NO3)3, (NH4)2HPO4, glucose, lactose, maltose, fructose, sucrose, urea, uric acid,ascorbic acid, oxalic acid, lactic acid, l-cysteine, l-glutathione, dopamine, and melamine were selected for thedetermination of selectivity of FeNPs. It is obvious from 2, CaCl2, ZnCl2, MgCl2, Al(NO3)3, (NH4)2HPO4, glucose, lactose,maltose, fructose, sucrose, urea, ascorbic acid, oxalic acid, andlactic acid were approximately same with the control experiment usingthe distilled water. Furthermore, as can be seen in 2, CaCl2, ZnCl2, MgCl2, Al(NO3)3, (NH4)2HPO4, glucose, lactose, maltose, fructose,sucrose, urea, ascorbic acid, oxalic acid, and lactic acid were between97.46 and 103.17%. These results revealed that the colorimetric AAdetection could be carried out successfully with FeNPs synthesizedfrom the hyperaccumulator plant of P. brutia in the presence of various interfering substances except for uricacid, l-cysteine, l-glutathione, dopamine, and melamine.The possible interfering substances such as NaCl, KCl, CuCl3.5P. brutia possessed the potentialto be applied accurately and reliably to the detection of AA contentin real samples.For the investigationof the reliability of the colorimetric AA detection with FeNPs, themethod was applied to analyze AA content in real samples. As presentedin 4Pinus brutia as a natural metal ion source to synthesize novel iron-based nanoparticlesfor the antibacterial activity and the colorimetric detection of ascorbicacid. The results showed that FeNPs exhibited a significant bactericidaleffect toward Gram-positive (L. monocytogenes and S. aureus) and Gram-negative(E. coli(O157: H7), E. coli(ATCC 25922), S. enteritidis, and S. typhimurium) pathogenic bacteria. The findingssuggested that the leach solution prepared from the iron hyperaccumulatorplant of Pinus brutia could be usedfor developing antibacterial FeNPs against pathogenic bacteria. Furthermore,a sensitive and selective colorimetric AA detection system was successfullyconstructed using FeNPs as enzyme mimics. This colorimetric detectionsystem using FeNPs could be applied to quantify AA concentration,with a linearity range of 30–200 μM and an LOD valueof 0.5462 μM. It was observed that the colorimetric AA detectioncould be effectively carried out in the presence of various interferingsubstances except for uric acid, l-cysteine, l-glutathione,dopamine, and melamine. It was also tested to quantify AA in realsamples, and their recovery values were 100.59–104.57% withRSD less than 4%, proving its feasibility in evaluating AA contentin practical application. Considering all of these, the present worknot only provides a novel antibacterial agent and a peroxidase-likecatalyst but also inspires researchers to further explore varioushyperaccumulator plants for the synthesis of metallic nanoparticlesfor a variety of applications.In summary, we first developeda new facile method using the leachsolution of hyperaccumulator plant


These SNPs were located in close proximity to or within the sequence of the thirteen candidate genes, such as Galanin And GMAP Prepropeptide (GAL), Centromere Protein (CENPF), Glypican 2 (GPC2), Phosphatidylethanolamine N-Methyltransferase (PEMT), Transcription Factor AP-2 Delta (TFAP2D), and Carboxypeptidase Q (CPQ) gene related to egg-laying and Solute Carrier Family 5 Member 7 (SLC5A7), Neurocalcin Delta (NCALD), Proteasome 20S Subunit Beta 2 (PSMB2), Slit Guidance Ligand 3 (SLIT3), and Tubulin Tyrosine Ligase Like 7 (TTLL7) genes related to egg quality. Interestingly, one of the genes involved in bone formation (SLIT3) was identified as a candidate gene for ESS. Our candidate genes and SNPs associated with egg-laying traits were significant for molecular breeding of egg-laying traits and egg quality in LingKun chickens.Egg production is the most important trait of laying hens. To identify molecular markers and candidate genes associated with egg production and quality, such as body weight at first oviposition (BWF), the number of eggs produced in 500 days (EN500), egg weight (EW), egg shell thickness (EST), egg shell strength (ESS), and Haugh unit (HU), a genome-wide analysis was performed in 266 LingKun Chickens. The results showed that thirty-seven single nucleotide polymorphisms (SNPs) were associated with all traits ( However, despite a range of studies in this area, wide confidence intervals (CIs) for the positions of QTL remain that have rarely been replicated. A new research era was initiated with advances in sequencing technology and single nucleotide polymorphism (SNP) chip, and genome-wide association study (GWAS) has become one of the most effective methods to detect genetic variation in livestock. In previous studies, two SNPs related to body weight at first oviposition (BWF) were located at 78.8 and 78.9 Mb, respectively, on the GGA4 gene by GWAS of Jinghai Yellow chicken, and both were located in FAM184B gene  contains 16,271 QTLs from 367 publications, which represent 442 different traits , and ESS and EST are significantly correlated (p = 0.555). Meanwhile, EST and ESS were negatively correlated with EN500. Meanwhile, Manhattan diagram (A) and QQ diagram (B) of correlation analysis results are shown in The basic statistics for egg-laying traits and egg quality in the Lingkun chicken for the GWAS are shown in p-value = 3.84E-09) was located on chromosome 3 at position 21,147,253 bp, which was found within the Centromere Protein (CENPF) gene, an associated SNP G16754641A  located on chromosome 5 at position 16,754,641 bp was found to be within the upstream of the Galanin And GMAP Prepropeptide (GAL) gene, two significant SNPs, G3321921T  and G3321930A , were located at 3,321,921 and 3,321,921 bp on chromosome 9, respectively, and both were within the Glypican 2 (GPC2) gene, and two significant SNPs, T4287370A  and C5246144G , were located at 4,287,370 and 5,246,144 bp on chromosome 14, respectively, which were found to be within the upstream of the Cytochrome P450  and Phosphatidylethanolamine N-Methyltransferase (PEMT) gene. An associated SNP G2854459T  was located on chromosome 23 at position 2,854,459 bp, which was found to be within the downstream of the Transmembrane Protein 200B (TMEM200B) gene.A total of eight significant loci distributed on chromosomes 3, 5, 6, 9, 14, and 23 were associated with BWF ; Table 3EN500. The most associated SNP C108380147G  was located on chromosome 3 at position 108,380,147 bp was found 62 kb downstream of the Transcription Factor AP-2 Delta (TFAP2D) gene. Seven significant SNPs were located within the 69.78 Mb segment (between 57.21 and 126.99 Mb) on chromosome 2. In between, two significant SNPs, A57924869T  and A57925378C , were located at 57,924,869 and 57,925,378 bp, respectively, and both were within the GPC2 gene, an associated SNP A57945258T  was located at position 57,945,258 bp, which was found within the Neurensin 1 (NRSN1) gene, two significant SNPs, C126998745A  and T126998774C  were located at 12,699,8745 and 126,998,774 bp and both were within the Carboxypeptidase Q (CPQ) gene.A total of seven significant loci distributed on chromosomes 2 and 23 were associated with p-value = 1.63E-08) and T137349076C , located at positions 137,349,071 and 137,349,076 bp, were found to be within the Solute Carrier Family 5 Member 7 (SLC5A7) gene. Four significant SNPs were located within 48.93 Mb segment (between 79.95 and 128.89 Mb) on chromosome 2. In between, 79958113G , C83033381T , and 128890264G  were located at 79,958,113, 83,033,381, and 128,890,264 bp, respectively, which were found to be within the downstream of Uridine Phosphorylase 1 (UPP1) gene, Formin Homology 2 Domain Containing 3 (FHOD3) gene, and Neurocalcin Delta (NCALD) gene. The associated SNP C17452066T  was located on chromosome 3 at position 17,452,066 bp, which was found within the Actin Regulator (ENAH) gene, an associated SNP T6405164C  located on chromosome 8 at position 6,405,164 bp was found to be within the Centrosomal Protein 350 (CEP350) gene, In addition, an associated SNP C8498988T  was located on chromosome 17 at position 8,498,988 bp, which was found within the Ring Finger Protein 2 (RNF2) gene.A total of fourteen significant loci distributed on chromosomes 1, 2, 3, 8, and 17 were associated with EW. Seven significant SNPs were located within 28.08 Mb segment (between 137.34 and 165.43 Mb) on chromosome 1, in between, two associated SNPs, C137349071T  was located on chromosome 23 at position 4,415,750 bp, which was found within the upstream of the Proteasome 20S Subunit Beta 2 (PSMB2) gene. A total of four SNPs  were significantly associated with ESS, which were located within the 3,769 kb segment  on chromosome 13, which were found within the Slit Guidance Ligand 3 (SLIT3) gene.Two significant SNPs were associated with EST, the most associated SNP C4415750T  was located on chromosome 13 at position 17,272,906 bp, which was found within the downstream of Tubulin Tyrosine Ligase Like 7 (TTLL7) gene.The associated SNP A17272906G . All phenotypic values of traits in genotyped individuals were tested for normality, and some abnormal values that were extremely deviating from normal distribution were deleted before conducting association tests.Blood samples were collected from the wing vein of 266 female offspring using heparin sodium as anticoagulants. The genomic DNA of each individual was extracted from Lingkun chicken using the E.Z.N.A. Tissue DNA Kit (Omega Bio-Tek) following the Tissue DNA – Spin Protocol. After the DNA samples were delivered, a quality control test was carried out on the specimens, and the qualified DNA  was used to do further study. All the samples in this experiment were tested using genomic DNA gelose gel electrophoresis and a total of 266 have reached the qualified requirements. Electrophoresis point holes were required to be clean and pollution-free, the main band was clear, and there was no trailing.ftp://ftp.ensembl.org/pub/release-76/fasta/.We chose Illumina pair-end sequencing (PE150) to do the resequencing project. For Illumina pair-end sequencing, at least 1 μg of genomic DNA was used for sequencing library construction for each sample. Paired-end libraries with insert sizes of ≈450 bp were prepared following Illumina's standard genomic DNA library preparation procedure. Purified genomic DNA is sheared into smaller fragments with the desired size by Covaris, and T4 DNA polymerase was applied to generate blunt ends. After adding an “A” base to the 3' end of the blunt-phosphorylated DNA fragments, adapters are ligated to the ends of the DNA fragments. The desired fragments can be purified through gel-electrophoresis, then selectively enriched and amplified by PCR. The index tag could be introduced into the adapter at the PCR stage as appropriate followed by a library quality test. After being quantified by TBS380, paired-end libraries were sequenced by Shanghai Biozeron Biotechnology Co., Ltd.  with the Illumina HiSeq platform according to the manufacturer's standard protocols. In this experiment, clean reads (72788874.44) were generated and 99.18% of these were mapped to the chicken genome −8 were rejected. Finally, 266 individuals and 9,327,106 SNPs were identified in the present study. The experiment fast structure (https://github.com/rajanil/fastStructure) was used to perform population structure analysis. It was based on the maximum likelihood method (MLM). In addition, a principal component analysis (PCA) for the independent 9,327,106 SNPs was performed in Genome-wide Complex Trait Analysis (GCTA) (version 1.24). In order to reduce the influence of population stratification, the first and second components of the PCA were used in the model.A total of 1,509,826,877 SNPs were obtained. Quality control of the genotyping data was performed in PLINK (v1.9). During this process, markers were selected based on three conditions: The SNPs that exhibited minor allele frequencies (MAFs) lower than 5%, low minor allele frequency lower than 2%, and a Hardy–Weinberg equilibrium tests lower than 1.0 × 10The general linear regression model (GLM) in PLINK was used in this study. The model is as follows:b is a vector matrix of fixed effects that includes population structure, g is a vector of SNP effects with p-value of the 5% Bonferroni genome-wide significance was calculated based on the estimated number of independent SNPs and LD blocks for autosome SNPs. The threshold p-value of the 5% Bonferroni genome-wide significance was set at 9.47E-08 , and the threshold p-value for significance was 1.89E-06 .where Y is the vector of observations; X is a design matrix of covariates containing all other fixed effects; The gene closest to the SNP with an extremely significant correlation is considered as a candidate gene. Sequence alignment was conducted through the National Center for Biotechnology Information (NCBI) and Ensembl databases to identify genes within 100 KB upstream and downstream of significant SNPs.https://bigd.big.ac.cn/gsa/browse/CRA006206).The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: National Genomics Data Center, China National Center for Bioinformation .LL, HC, SZ, YZ, SL, CW, YT, and TZ contributed to conception and design of the study. XL organized the database. WX performed the statistical analysis. JG wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.The authors are grateful for the financial support provided by Zhejiang Major Scientific and Technological Project on Agricultural (Livestock's) Breeding (2021C02068-10) and Wenzhou New Poultry Variety Breeding Cooperation Group Project (2019ZX005).XL was employed by the company Wenzhou Golden Land Agricultural Development Co., Ltd. The remaining 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.


The authors wish to correct the following error in this paper .Text CorrectionIt was stated that 73 articles were included, comprising 30 investigations for OCD DBS patients, the manuscript has been corrected to 71 articles comprising 28 investigations for OCD DBS patients. Therefore, 47 (not 45 at stated) articles were excluded; 11 (not 9 as stated) were excluded for reporting of the primary outcomes in another article, resulting in 9 (not 10 RCTs as stated), and a total 153 (not 181 as stated) OCD DBS cases. The graphical abstract has been updated accordingly, please see Figure 1.There were errors in the original publication. 3.7 DBS Results:A correction has been made to n = 16), reporting of primary outcomes in another article (n = 11), lack of pre- to post- operative outcomes (n = 5), adjunct therapy having been implemented (n = 4), the primary diagnosis not being an OCRD or was unclear (n = 4), previous DBS for Parkinson’s (n = 1), or presence of comorbid psychosis (n = 1). OCD investigations included 9 RCTs (six with an open-label extension), five open-label trials, two follow-up reports, one pilot study, seven case series, and four case reports. TS investigations included three RCTs with open-label extension, five open-label trials, five follow-up reports, one pilot study, three retrospective reports, seven case series, and 17 case reports. BDD investigations included a single case study. The final sample included, 153 OCD patients, 175 TS patients, and one BDD patient.One hundred and eighteen articles were screened for eligibility: 71 were included in the final synthesis, comprising 28 investigations for OCD, 42 for TS, and 1 for BDD. Forty-seven articles were excluded due to a lack of standardized assessment of primary symptoms , and the outcomes relating to the BNST DBS target.3.7.1 DBS Results for OCD:A correction has been made to One RCT implanted two targets (four electrodes) per patient; DBS of the VC/VS, amSTN, and both targets achieved 53%, 45% and 60% mean improvement, respectively [168]. Two open-label studies compared two targets: NAc DBS led to 12%–23% improvement, where-as BNST DBS led to 24–39% improvement .Investigations of NAc DBS included three RCTs, one open-label trial, and three case reports. RCTs led to 13% and 51% symptom improvement , and long-term treatment (6–12 months) led to 12–33% improvement .Investigations of ALIC DBS encompassed two RCTs, one trial with a staggered switch on, one long-term follow-up report, and two case reports. Closed-label investigations led to 20% and 43% improvement ; and long-term (1–9 years) treatment led to 43–67% improvement . The ALIC was also targeted in the cohort of Mantione et al., (2014) through shifts in targeting, and achieved 43% improvement at 1 year [220].Investigations into VC/VS DBS involved one RCT, one open-label trial with a long-term follow-up report, and three case reports. The RCT originally implanted the ALIC [153], and implemented a posterior shift in target to the VC/VS. A larger cohort from the same site as Nuttin (2003) achieved 42% mean improvement from closed-label treatment, and at three-year follow up, 39% symptom improvement was maintained [156].Investigations of amSTN DBS involved a multisite RCT that resulted in 25% median improvement, and 51% mean improvement was reached at four-year follow up [158].DBS of the ITP, slMFB, and thalamus were also targeted for OCD, 52%, 42%, and 9% mean change was respectively achieved per target .The manuscript stated that the RoB assessment for OCD DBS articles rated 47 as low risk, 17 as medium risk and nine as high risk. The manuscript has been revised to include 46 as low risk, and 16 as medium risk. The quality assessment for OCD DBS articles rated 46 as moderate, the manuscript has been revised to include 44 as moderate. The manuscript reported that 18 (9.9%) OCD patients had their devices switched off, the proportion of patients has been revised to 11.7%. Lastly, it was reported that comorbidities in OCD DBS patients were not reported in 72 cases, this has been amended to include 44 cases.3.8 DBS Discussion:A correction has been made to The RoB assessment rated 46 articles as low risk, 16 as medium risk, and nine as high risk (S2). The quality assessment rated 19 articles as good, 44 as moderate and eight as poor (S3). Only 11 out of 71 articles were RCTs, and 35 were case reports, which meant a randomized control aspect and group level analysis was not present in almost half of the patients included here. Furthermore, only half (36) of the articles reported on more than one time-point, which limits interpretations regarding the duration and pattern of response. Within the bias assessment, there were multiple deviations from the intended protocol, including DBS explants or switch off, and closed-label conditions ending early. It was reported that 18 (11.7%) OCD patients and 12 (6.8%) TS patients had their devices switched off or explanted due to limited/no efficacy or even worsening in some instances; a further three (1.7%) TS patients underwent repositioning. Also, five RCTs had patients that ended the closed-label phase early. It is possible that not all cases of device switch off, explant, or repositioning were captured.Adverse events included transient psychiatric symptoms, particularly hypomania, increased anxiety, deterioration of mood and suicidal thoughts, which were generally resolved with programming adjustments. There were seven suicide attempts, and one completed suicide [157]. Battery depletion was rarely reported on but seemed to occur between 5–22 months in OCD cohorts  and was reported to occur at 24-months for one TS patient [210].n = 17), personality disorder (n = 4), bipolar (n = 3), PD (n = 2), GAD (n = 1), panic disorder (n = 1), BDD (n = 1), TS (n = 1), yet was most often not reported (n = 44). Comorbidities reported in TS trials included OCD (n = 37), MDD (n = 29), ADHD (n = 15), GAD (n = 5), dystonia (n = 3), panic disorder (n = 1), personality disorder (n = 1), and was not reported in a further 26 cases.There was large heterogeneity in protocols, and no comparable protocols were identified, making a meta-analysis not possible. To elaborate, surgical procedure, target trajectory, programming method, stimulation location, comorbidities, follow-up duration, and closed-label conditions varied greatly. Comorbidities reported in OCD trials included MDD  was considered a distinct cohort to the article by Greenberg et al., (2010), and the article by Liebrand et al., (2019) was considered a distinct cohort to the article by Mantione et al., (2014); however, the cases are in fact the same, thus the article by Suetens et al., (2014) and Liebrand et al., (2019) have been removed from  All the reference number have been updated accordingly.In the original publication, there was a mistake in Table 12 appears below.The corrected The authors apologize for any inconvenience caused and state that the scientific conclusions are unaffected. The original publication has also been updated.


In addition, we highlight similar pathogenic mutations in genes encoding a specific regulatory subunit of a multimeric PSP. Overall, we describe 19 affected genes, and find that most pathogenic variants are loss-of-function, with just a few examples of gain-of-function alterations. Moreover, despite their widespread tissue expression, the large majority of congenital PSP disorders are characterised by brain-specific abnormalities, suggesting a generalized, major role for PSPs in brain development and function. However, even if the pathogenic mechanisms are relatively well understood for a small number of PSP disorders, this knowledge is still incomplete for most of them, and the further identification of downstream targets and effectors of the affected PSPs is eagerly awaited through studies in appropriate in vitro and in vivo disease models. Such lacking studies could elucidate the exact mechanisms through which these diseases act, and possibly open up new therapeutic avenues.Reversible protein phosphorylation is a fundamental regulation mechanism in eukaryotic cell and organismal physiology, and in human health and disease. Until recently, and unlike protein kinases, mutations in serine/threonine protein phosphatases (PSP) had not been commonly associated with disorders of human development. Here, we have summarized the current knowledge on congenital diseases caused by mutations, inherited or  Reversible protein phosphorylation lies at the core of protein regulation, fine-tuning their activity and stability in cells and tissues. Through addition or removal of a negatively charged phosphate group, protein activity can be up- or downregulated, or their stability promoted or inhibited. In homeostatic conditions, a delicate phosphoproteomic balance is achieved through the counterbalanced action of phosphorylating kinases, and dephosphorylating phosphatases. Consequently, when this balance is disrupted, disease inevitably follows, making kinases and phosphatases prime therapeutic targets in cancer, neurodegenerative diseases, diabetes, cardiac diseases, immune dysfunction, development, and other human pathologies. As protein kinases were discovered several decades ahead of protein phosphatases, the latter enzymes generally lag a bit behind their enzymatic antagonists, both in terms of general understanding of their function and regulation, and in terms of their implication or therapeutic targeting in human disease .Regulatory phosphorylation occurs on either a serine, threonine, or tyrosine residue. Due to tyrosine having a benzene ring, and the similar structure between serine and threonine, two major classes of protein phosphatases have been identified, the serine/threonine phosphatases (PSP), and the protein tyrosine phosphatases (PTP). In this review, we will focus on the serine/threonine phosphatase family of proteins, highlighting their roles in inborn genetic disorders.PSP phosphatases specifically dephosphorylate phospho-Ser or phospho-Thr residues, which, throughout the proteome, represent the large majority of protein phosphorylation, with phospho-serine estimated 86.4% and phospho-threonine 11.8% . In humaThe subfamily of phosphoprotein phosphatases (PPP) are further subdivided into seven different classes, PP1 through PP7 , all of 2+/calmodulin, PP3 becomes active  to form large and unique interaction interfaces with PP1  which are highly similar but differ in substrate specificity and activity , the pleckstrin homology domain leucine-rich repeat protein phosphatases (PHLPP), and the pyruvate dehydrogenase phosphatases (PDPs). All these phosphatases share a conserved catalytic core region, with highly conserved amino acid sequences responsible for phosphate- or metal-binding  Shi, 20. The PP2activity . The PHL subunit   sharing a conserved structural organisation in the catalytic core. Here again, highly conserved amino acids within this family of phosphatases bind metal-ions and phosphates necessary for phosphatase activity , and a transcription factor interacting helix (TFIIF), important in substrate binding and thus phosphatase activity , and in part through emerging insights into the role of  disease . These adiseases .Recently, many new initiatives have been taken to focus on deciphering until then unknown causes of disease. A prime example is the Deciphering Developmental Disorders (DDD) study in the United Kingdom, having screened over 33,000 parents and children as of January 2020, leading to the identification of 67 completely new disorders, including several involving phosphatase genes . This exIn this review, we aim to create an overview of currently known, inborn genetic diseases caused by mutations in PSP phosphatase genes. Thus, we aim to provide a reference framework to further study these novel genetic disorders, and spur advancement in mechanistic insight in these diseases.PPP1CB has been defined as the cause of a congenital disease, i.e. Noonan-like syndrome with loose anagen hair-2. In addition, three major PP1 regulatory subunit genes also confer inborn disease upon mutation: PPP1R12A, PPP1R15B, and PPP1R21.PP1 phosphatases form a myriad of holoenzyme complexes, with a diverse set of regulatory subunits or PIPs . This wain vivo in memory formation . Typically, this disorder is caused by mutations in SHOC2, encoding a leucine-rich repeat scaffold protein, which in complex with PP1-Cβ and MRAS, activates the RAS/MAPK pathway  mutations in PPP1CB have been implied as causative of intellectual disability and congenital heart disease. Ma et al. identified eight individuals with heterozygous, de novo variants in PPP1CB, presenting with intellectual disability, developmental delay, congenital heart disease, macrocephaly, short stature, and dysmorphic features , which forms a complex with PP1 catalytic subunit Cβ and M20/21 to form myosin phosphatase. This holoenzyme regulates function of non-muscle myosin II, and the interaction between myosin and actin in smooth muscle cells, through the phosphorylation state of the myosin light chain - with dephosphorylation of the light chain activating myosin function. MYPT1 is involved in embryogenesis by regulating cell adhesion and motility in the eye of Drosophila, embryonic elongation in C. elegans, gastrulation in D. rerio, and embryonic viability after 7.5 days post conception in mice , presenting with a neurological phenotype, urogenital malformations, or an overlap of both (618820). Furthermore, a discordance between genotypic and phenotypic sex was observed in two individuals. The neurological phenotype includes, but is not limited to, brain malformations such as holoprosencephaly, macro- and microcephaly, dysmorphic facial features, seizures and a high frequency of developmental delay. Nine individuals presented with urogenital malformations, including malformations of the genitalia in both sexes, such as the presence of a uterus in XY individuals. No clear genotype-phenotype association could be established, given the wide diversity of phenotypic features, and the variants being spread among multiple exons and domains , is involved in protein kinase R-like endoplasmic reticulum kinase (PERK) signalling. PERK itself phosphorylates eukaryotic translation initiation factor 2α (eIF2α), thus downregulating protein translation . The Arg658Cys substitution is located in the conserved C-terminal functional core of the protein, near the RVxF-motif , characterized by neonatal diabetes associated with skeletal dysplasia and/or episodes of acute liver failure  (OMIM#619383). The first study identified three patients with homozygous null variants in PPP1R21, encompassing two homozygous nonsense variants , and one homozygous frameshift variant (p.Gly30Cysfs*4)    . AnotherCysfs*4) . Most re., 2019;  (OMIM#61Cysfs*4) . All pat., 2019;  , and the variable B-subunits, belonging to four different families, are encoded by 15 distinct genes: PPP2R2A, PPP2R2B, PPP2R2C, PPP2R2D ; PPP2R3A, PPP2R3B, PPP2R3C ; PPP2R5A, PPP2R5B, PPP2R5C, PPP2R5D, PPP2R5E ; and PPP2R6A, PPP2R6B and PPP2R6C . This diversity in subunits results in more than 100 possible PP2A trimers, in which the B-subunit confers substrate specificity and localisation, the C-subunit catalytic activity, and the A-subunit structural integrity , PPP2CA (Cα), PPP2R2C (B55γ), PPP2R5B (B56β), PPP2R5C (B56γ), and PPP2R5D (B56δ), with most patients and mutations, so far, found in PPP2R5D (https://jordansguardianangels.org/). In contrast, mutations in PPP2R3C (encoding the regulatory B″γ or G5PR subunit) were found to be mainly associated with deficits in gonadal development (PPP2R2B (encoding B55β) with spinocerebellar ataxia . Patients presented with a heterogeneous set of symptoms, including mild to severe intellectual disability and developmental delay, autism spectrum disorder, seizures and brain abnormalities . Here, patients can be classified into at least two clinical subsets, one milder and one more severe group. Both groups display typical PP2A-related neurodevelopmental symptoms such as hypotonia, intellectual disability and developmental delay. However, the mild subgroup of individuals tends to have macrocephaly, no epilepsy, and sometimes, microtia and loss of hearing, while patients in the severe subgroup tend to present with microcephaly, seizures, facial dysmorphisms, corpus callosum hypoplasia or agenesis, and sometimes, congenital heart defects  and less common symptoms, such as epilepsy, autism spectrum disorder, ADHD and early-onset Parkinsonism (616355). Thus far, seventeen variants have been reported for PPP2R5D which are spread throughout the whole protein, but some seem to cluster around a conserved acidic loop, or a canonical short linear interaction motif-binding site  leading to a constitutive activation of the Akt-mTOR pathway when reconstituted in HEK293 cells  abnormalities, and often, epilepsy and ASD. Despite being relatively well characterised molecularly, an exact cellular disease mechanism remains unknown, in terms of which neuronal substrates could be affected in their (de)phosphorylation. Thus, to fully understand these diseases, more research needs to be performed to pinpoint which molecular pathways are affected by variants in PP2A subunits. Due to the lowering costs of genomic sequencing tools, more patients are being identified worldwide, and additional variants are found that could help expand the current knowledge regarding molecular and biochemical properties of PP2A subunits.PPP2R2B (OMIM#604326) and PPP2R3C (OMIM#618419)  .PPP2R2B, encoding the B55β subunit, was implicated as causative in spinocerebellar ataxia 12, with patients presenting with upper extremity tremor, progressing to head tremor, gait ataxia, dysmetria, dysdiadokinesis, hyperreflexia, paucity of movement, and abnormal eye movements - eventually developing dementia. Patients were found to be carrying a number of expanded CAG repeats, 133 nucleotides upstream of the transcription start site of PPP2R2B . PPP2R3C, encodes the Ca2+-regulated B″γ/G5PR subunit and is relatively ubiquitously expressed , one Ca2+-binding regulatory subunit (PPP3R1 or PPP3R2), and Ca2+/calmodulin to release auto-inhibition   forms a complex of one catalytic subunit  , while Ptibility . Calcinetibility . In the tibility . Embryontibility . Congeniet al. found several mutations, generally mapping to two distinct locations in the protein , making them intolerable to high calcium growth conditions. In this model, overexpression of ppb1 with the conserved mutations in the catalytic domain failed to rescue this growth phenotype , PPM1F, and PPM1K (OMIM#615135). PPM1B is one of the genes lost in the homozygous 2p21 deletion syndrome  is highly expressed in adult brain and gastrointestinal tissue, while PPM1B RNA is found in high quantities in muscle tissue , encoded by oncogene . PPM1D R tissues . Furthere effect .PPM1D in brain tissue, it came as no surprise that mutations in PPM1D can cause disease related to brain development. De novo, heterozygous mutations in PPM1D have indeed been identified as the cause of Jansen–de Vries syndrome, with patients presenting with mild to severe intellectual disability and/or developmental delay, autism spectrum disorder, ADHD, anxiety disorders, hypotonia, broad-based gait, facial dysmorphisms, and periods of fever and vomiting , probably barring the protein from entering the nucleus, while the wild-type protein still does. WIP1 plays an important role in cellular stress, notably in the p53 pathway following DNA damage  - hence its frequently used protein name, CAMK phosphatase . As one of two possible catalytic subunits of pyruvate dehydrogenase phosphatase, PDP1 is predominantly expressed in skeletal muscle, while PDP2 is more abundant in liver. PDP1 localises to the mitochondrial matrix, where it catalyses the dephosphorylation of pyruvate dehydrogenase, thus activating it  only the first one is currently known to cause an inborn genetic disease (OMIM#604168)  syndrome  . CTDP1 eitiation , and is itiation . A singlsyndrome . CCFDN sof CTDP1 . Thus, ted decay . Interesde novo mutations occurring during gametogenesis or post-zygotically, can become a major cause of developmental disorders  were not commonly associated with disorders of human development. Overall, of 38 human genes encoding PSP catalytic subunits or monomeric phosphatases, 10 were found to be affected in congenital disorders or were associated with an increased disease predisposition: PPP1CB, PPP3CA (some) and PPP2R3C). This observation has important ramifications for potential therapeutic interventions, as in general, it is more difficult to restore damaged phosphatase activity or expression by pharmacologic modulators, than to inhibit uncontrolled phosphatase activity or to interfere with increased phosphatase-substrate binding. In the former cases, alternative targeting of the relevant PSP downstream targets, e.g. by interfering with the upstream protein kinase activities, remains an appealing option to compensate for the loss of PSP activities. However, this necessitates knowledge about the affected physiologic substrates of the phosphatase, dysphosphorylation of which is causally involved in certain disease symptoms, as well as knowledge about the identity of the upstream kinase(s), and these insights are still often lacking. On the other hand, several PSP phosphatase activating pharmaca or peptide mimics did recently emerge . Moreover, it brings up the provocative question how different variants within a single affected PP2A gene, and variants in different PP2A genes may, or may not, relate to each other and affect similar pathways, potentially resulting in common, or different, molecular and/or clinical subgroups. The latter may have, again, important consequences for therapeutic intervention strategies.In general, the functional impact of identified mutations is easier to understand and to investigate in the monomeric PSP phosphatases, while this can be much more complex in the multimeric phosphatases, especially if mutations affect the common catalytic C-subunit , and thus may have different effects on different holoenzyme complexes. For example, extensive molecular characterisation of Remarkably, in this overview of PSP congenital disorders, we often have encountered phenotypes that are specifically associated with brain dysfunction or dysmorphology, despite mutations being omnipresent in the human body, and the widespread tissue expression of most PSP genes. Thus, we must conclude that PSP-dependent protein dephosphorylation has a major potential for regulation of brain development and function. Nevertheless, some PSP disorders manifested themselves in other organs too, including the (uro)genital and endocrine systems, and were, in these cases, often linked to alterations in specific molecular pathways.PPP1CB, PPP1R15B, PPM1K and PDP1, followed by the PPP2- and PPP3CA-related neurodevelopmental disorders. Even in these relatively better understood diseases, knowledge is still incomplete, and in particular, the further identification of downstream targets and effectors could elucidate the exact mechanisms through which these diseases act, and possibly open up therapeutic avenues. To this end, new emerging technologies to identify PSP phosphatase substrates have been proposed that could be of value  these gene variants cause the associated clinical phenotypes.


The sirtuin SIRT6 is predominantly located in the nucleus and shows deacetylase activity for acetylated histone 3 lysine 56 and lysine 9 as well as for some non-histone proteins. Over the past decade, experimental analyses in rodents and non-human primates have demonstrated the critical role of SIRT6 in extending lifespan. Recent studies highlighted the pleiotropic protective actions of SIRT6 in angiogenesis and cardiovascular diseases, including atherosclerosis, hypertension, heart failure, and stroke. Mechanistically, SIRT6 participates in vascular diseases via epigenetic regulation of endothelial cells, vascular smooth muscle cells, and immune cells. Importantly, SIRT6 activators  have provided therapeutic value for treating age-related vascular disorders. Here, we summarized the roles of sirtuins in cardiovascular diseases; reviewed recent advances in the understanding of SIRT6 in vascular biology, cardiovascular aging, and diseases; highlighted its therapeutic potential; and discussed future perspectives.Aging is a key risk factor for angiogenic dysfunction and cardiovascular diseases, including heart failure, hypertension, atherosclerosis, diabetes, and stroke. Members of the NAD Various diseases are correlated with aging, including cardiovascular diseases, stroke, cancer, and chronic obstructive pulmonary disease ,2. For iIn recent decades, numerous pivotal regulators of aging have been identified, including sirtuins, mammalian target of rapamycin, insulin-like growth factor (IGF), AMP-activated protein kinase, and forkhead box O (FoxO) transcription factor family -11. Thes2.+-dependent class III histone deacetylases with deacetylase and ADP-ribose transferase activities [The sirtuin family comprises NADtivities -15. In mtivities  Fig. 1)+-dependetivities . In additivities ,18. ThusSirtuins are enzymes that slow the process of aging and aging-related disorders across species. Genetic depletion of SIRT1, SIRT6, and SIRT7 affects the development and reduces the lifespan of mice, accompanied by cardiovascular defects -21. Althvia activation of liver kinase B1-AMP-activated protein kinase signaling and contributes to metformin-mediated anti-hypertrophic effects [The roles of sirtuins in cardiovascular diseases have been widely studied over the past decade. In general, sirtuin family members, except for SIRT4, are cardioprotective factors . SIRT1 h effects . The car effects ,32. SIRT effects ,34. Furt effects .SIRT3-mediated metabolic homeostasis in mitochondria also contributes to its cardioprotective functions ,37. InteInterestingly, sirtuins play important roles in cardiac tissues in a deacetylase-independent manner. For instance, we observed that the effects of SIRT4 on MnSOD and mitochondrial ROS in hypertrophic cardiomyocytes did not rely on its enzymatic activity . Moreovein vivoroles of other sirtuins in vascular biology and aging-related vascular diseases remained largely unknown. Over the past two years, the understanding of SIRT6 in vascular diseases has achieved remarkable progress.The roles of SIRT1 and SIRT3 in vascular biology have been widely studied . SIRT1 i3.Among the sirtuin family members, SIRT6 is a key regulator of genome stability, stemness, and aging. SIRT6 deficiency reduces the lifespan of mice ,60,61. IThe role of stem cells in aging is critical, and some studies have focused on the functions and mechanisms underlying SIRT6 in stem cells. For instance, Pan et al.  reported4.Senescence, phenotype switching, and activation of vascular cells and immune cells are hallmarks of vascular aging and contribute to vascular diseases, including atherosclerosis, hypertension, stroke, arterial aneurysm, and vascular injury in myocardial infarction. The critical role of SIRT6 in regulating aging and stem cell senescence has driven studies of SIRT6 in vascular aging and diseases. Recent studies from our lab and others revealed that SIRT6 protects against vascular aging. SIRT6 is widely expressed in vascular cells and participates in vascular biology by epigenetically regulating endothelial cells (ECs), VSMCs, and immune cells. Here, we discuss recent advances in the understanding of SIRT6 in aging-related vascular diseases, including atherosclerosis, hypertension, and ischemic stroke.4.1-/- mice fed a high-fat diet (HFD), endothelial loss of SIRT6 promotes monocyte adhesion to ECs, impairs endothelium-dependent vasorelaxation, and facilitates atherosclerosis development [Endothelial dysfunction and senescence are the initial steps in atherosclerosis development. SIRT6 is a well-known regulator of endothelial senescence. As discussed previously, SIRT6 is essential for DNA damage repair and genome stability in stem cells and plays a similar role in vascular cells. SIRT6 deficiency in human ECs increases DNA damage, the formation of telomere dysfunction-induced foci, and the fraction of senescent cells as well as reduces cell proliferation and angiogenesis capacity . In humaelopment ,79. Thiselopment ,78. Collelopment ,81. Patielopment , revealielopment . Thus, t-/- mice by reducing the levels of H3K9ac and H3K56ac. This reduction represses the expression of ligands for natural killer (NK) group 2D (NKG2D), which is critical for activating NK cells to favor atherosclerosis development and plaque instability  and repressed its expression. Nkx3.2 is a transcription factor that controls the expression of GATA5, a novel regulator of blood pressure . Endothein vivo validation. The cardiovascular complications of SARS-CoV-2 infection include endothelial dysfunction and activation of immune cells, such as neutrophils [Angiotensin-converting enzyme 2 (ACE2) is a component of the renin-angiotensin-aldosterone system, the most important regulator of vascular aging and the pathophysiology of hypertension . In hypetrophils ,95. Becatrophils , aging-iVSMC senescence is an essential mechanism in vascular remodeling during the development of hypertension. A recent study demonstrated SIRT6 represses VSMC senescence . This meviaa similar mechanism. Thus, SIRT6 is an ideal target for treating atherosclerosis and hypertension because the roles of SIRT6 in each type of vascular cell protect the vascular tissues.These studies highlight the pivotal role of SIRT6 in aging-related cardiovascular disorders such as atherosclerosis and hypertension. The results also suggest that SIRT6 can prevent other types of vascular diseases, such as diabetic angiopathy, artery dissection, and ischemic vascular injury 4.2via epigenetic regulation or not) and whether AKT is critically involved in SIRT6 function in ischemic stroke. A previous study of the heart revealed that SIRT6 repressed AKT [Although recent studies have focused on SIRT6 in cardiovascular disorders, the role of SIRT6 in aging-related cerebrovascular diseases is not fully understood. Liberale et al. recently reported that endothelial SIRT6 preserved blood-brain barrier integrity and reduced stroke size by repressing endothelial apoptosis through AKT activation  Fig. 3CFig. 3C. ssed AKT , whereasssed AKT , suggestvia epigenetic mechanisms [viacell types other than ECs should be further evaluated.The mechanisms underlying SIRT6 functions in cardiovascular tissues significantly differ from those of its family member SIRT1. Generally, SIRT1 targets non-histone proteins in vascular cells, whereas SIRT6 regulates vascular cells chanisms ,88,90,97chanisms . Therefo5.SIRT6 exerts anti-aging roles in vascular tissues, indicating the therapeutic potential of SIRT6 activators. Importantly, SIRT6 in all cell types can repress cardiovascular aging and diseases, suggesting SIRT6 as an ideal target for treating aging-related cardiovascular diseases. Therefore, studies are needed to identify SIRT6 activators. Several SIRT6 activators have been identified in recent years, with some candidates showing the therapeutic value , as descviabinding to an allosteric site [via the zeste homolog 2/FOXC1 axis [Recent studies have provided a model for screening SIRT6 activators  for treating cancer, ischemic stroke, and other aging-related diseases such as liver fibrosis. In 2018, Huang et al. identified MDL-800, MDL-801, and MDL-811 as SIRT6 activators that increased SIRT6 deacetylase activity by up to 22-fold ric site . MDL-800ric site -102. SIRric site ,98,103. ric site . The novXC1 axis , highligXC1 axis , revealiTaken together, the currently identified SIRT6 activators MDL-800/811 show significant value as candidate drugs for treating aging and aging-related cardiovascular diseases. As SIRT6 controls the lifespan of non-human primates , the eff6.SIRT6 is critical for mammalian development and determines the lifespan of rodents and non-human primates. Findings obtained in the past two years have highlighted the vital role of SIRT6 in preventing angiogenic defects, vascular diseases, atherosclerosis, hypertension, and ischemic stroke. The anti-senescence function is crucial for the protective roles of SIRT6 in aging and vascular diseases. Importantly, recent studies suggested the translational value of SIRT6 activators, such as MDL-800/811. However, some functions of SIRT6 need further addressing.(1)Recent studies have mainly focused on the function of SIRT6 in the epigenetic regulation of cell senescence and inflammation. Cardiovascular diseases are closely related to metabolic syndrome. Accumulating studies have shown that SIRT6 mediates liver and adipose metabolism ,106, whi(2)e.g., miR-25-3p, LncRNA Blnc1, and LncRNA SNHG12) in vascular cells in vitro [Increased methylation of the SIRT6 gene and decreased SIRT6 protein stability occur in vascular injury. Some studies suggested that SIRT6 is regulated by miRNAs and lncRNAs SIRT6 is an anti-aging factor in rodents and primates ,63,67; t