Our previous study found that L-fucose significantly ameliorated DSS-induced colonic inflammation and intestinal epithelial injury. In this study, we discovered enhanced ISC regeneration and increased intestinal IL-22 secretion and its related transcription factor AHR in colitis mice after L-fucose treatment. Further studies showed that L-fucose promoted IL-22 release from CD4+ T cells and intestinal lamina propria monocytes (LPMCs) via activation of nuclear AHR. The coculture system of LPMCs and intestinal organoids demonstrated that L-fucose stimulated the proliferation of ISCs through an indirect manner of IL-22 from LPMCs via the IL-22R-p-STAT3 pathway, and restored TNF-α-induced organoid damage via IL-22-IL-22R signaling. These results revealed that L-fucose helped to heal the epithelial barrier by accelerating ISC proliferation, probably through the AHR/IL-22 pathway of LPMCs, which provides a novel therapy for IBD in the clinic.The recovery of the intestinal epithelial barrier is the goal for curing various intestinal injurious diseases, especially IBD. However, there are limited therapeutics for restoring intestinal epithelial barrier function in IBD. The stemness of intestinal stem cells (ISCs) can differentiate into various mature intestinal epithelial cells, thus playing a key role in the rapid regeneration of the intestinal epithelium. IL-22 secreted by CD4 Inflammatory bowel disease (IBD) is a nonspecific autoimmune disorder with intestinal mucosal damage as the main pathological manifestation, mainly including ulcerative colitis and Crohn’s disease ,2. Altho+ ISCs  at 37 °C and shaken at 150 rpm for 20 min. Subsequently, the fragments were cut into small pieces and digested in lamina propria digestive solution  at 37 °C and shaken at 150 rpm for 15 min. Afterwards, the supernatant was filtered with a 70 μm cell strainer, and the filtrate was centrifuged at 150 rpm for 5 min and washed with RPMI medium supplemented with 5% FBS . After centrifugation, the cells were resuspended in 40% Percoll solution (in PBS), overlaid on 80% Percoll solution, and then centrifuged at 500× g for 20 min at 20 °C. After spinning, the interface containing the LPMCs was aspirated and washed in medium for subsequent experiments. For in vitro experiments, LPMCs were divided into control, L-fucose (5 mg/mL) and L-fucose (5 mg/mL) +CH223191 (3 μM) groups as before and cultured in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin for 24 h, and a leukocyte activation cocktail  was added to the medium in the last 6 h.The small intestinal lamina propria monocytes (LPMCs) were harvested according to a previous study with a slight modification . BrieflyFor coculture experiments, the isolated LPMCs were cultured with intestinal crypts at a ratio of 20:1 in Matrigel according to previous work, though with a slight modification . The eff+ T cells and intestinal LPMCs were stained with fluorescent mouse antibodies specific for AF-700-CD45  and FITC-CD4  at 4 °C for 30 min. Then, the cells were fixed and permeabilized with fixation/permeabilization solution  for 30 min and washed with PBS. Next, LPMCs were stained with PE-IL-22 antibody  and BV421-ROR-γt antibody , and the CD4+ T cells were stained with PE-IL-22 antibody  at 4 °C for 30 min. After washing three times with PBS, the cells were analyzed by FACS . Data were analyzed by FlowJo V10 software .After the intervention, the CD4The measurement of organoids was performed according to a previous description . Briefly+ cells in per organoid were analyzed by ImageJ software.Cell proliferation in organoids was assessed by the BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 594  according to the manufacturer’s instructions. Briefly, the organoids were treated with 10 μM of EdU at 37 °C for 2 h and then fixed in 4% paraformaldehyde at room temperature for 15 min. After permeabilization with 0.3% Triton X-100, the organoids were reacted with reaction mixture for 30 min. Subsequently, the nuclei were stained with DAPI for 15 min. The EdU-stained 3D organoids were visualized under a Zeiss 710 Laser Scanning confocal microscope . The numbers of EdUFor immunohistochemical staining of Ki67, the terminal colon specimens were fixed with paraformaldehyde, embedded in paraffin and sectioned as described previously . First, For immunofluorescent staining, after the same deparaffinization and antigen retrieval steps mentioned above, the slides were permeabilized with 0.3% Triton X-100 at room temperature for 20 min, followed by blocking with 10% donkey serum for 1 h. Then, the sections were incubated with anti-mouse Lgr5 antibody  and anti-mouse IL-22R antibody  at 4 °C overnight. For pSTAT3 staining of organoids, the anti-mouse pSTAT3 antibody  was used as the primary antibody. The next day, the samples were then incubated with donkey anti-rabbit Alexa Fluor 488 , donkey anti-mouse Alexa Fluor 488  or donkey anti-rat Alexa Fluor 594  at room temperature for 1 h, followed by incubation with an anti-fluorescence quencher with DAPI . Fluorescence images were viewed and collected under a Zeiss 710 Laser Scanning confocal microscope.After treatment of the coculture system of LPMCs and organoids, the culture medium was collected and stored at −20 °C for ELISA analysis of TNF-α. Colonic specimens were collected from euthanized mice, homogenized and centrifuged, and the supernatant was stored at −20 °C for ELISA analysis of IL-22. A Mouse Interleukin 22 (IL-22) ELISA Kit  and Mouse Tumor necrosis factor α ELISA Kit  were utilized to measure the protein concentrations of IL-22 and TNF-α proteins level according to the manufacturer’s instructions. The absorbance was obtained with a microplate reader  at the corresponding nanometer wavelength.Both colonic tissues and intestinal organoids were harvested after treatment. Briefly, total RNA was isolated with TRIzol . Complementary DNAs (cDNAs) were obtained by reverse transcription using PrimeScript™ RT Master Mix . Gene expression was detected through amplification of cDNA fragments with SYBR Premix Ex TaqTM (TaKaRa) on a Roche LightCycler R480 . The relative expression levels of genes were normalized to the housekeeping gene GAPDH. All mRNA primer sequences are listed in v/v) phenylmethyl sulfonyl fluoride (PMSF), 1% (v/v) protease inhibitor and 1% (v/v) phosphatase inhibitor. Nuclear protein extraction from colon tissues and lymphocytes was performed according to the manufacturer’s instructions of the nuclear protein extraction kit . The bicinchoninic acid (BCA) assay kit  was used to detect protein concentrations. Equal amounts of denatured protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electrophoretically transferred to PVDF membranes . After blocking with 5% skimmed milk in TBS buffer containing 0.1% Tween-20, the membranes were incubated with rabbit anti-Lgr5 , rabbit anti-IL-22 , rabbit anti-β-catenin , rabbit anti-STAT3 , rabbit anti-phospho-STAT3-Y705 , rabbit anti-AHR  and rabbit anti-CYP1A1  antibodies. The mouse anti-β-actin  and rabbit anti-Lamin b1  antibodies were used for normalization. Afterwards, the membranes were incubated with goat anti-rabbit secondary antibodies  and goat anti-mouse secondary antibodies . The protein bands were visualized using an enhanced chemiluminescence (ECL) kit  under an Image Reader LAS-4000 imaging system .Western blotting analysis was performed according to a previous description . Brieflyt test; one-way ANOVA was employed to determine significant differences among multiple groups. p < 0.05 was considered statistically significant. SPSS 22.0  and Graphpad Prism software  were used for data analysis.All data are reported as the mean ± standard deviation (mean ± SD). Two groups were analyzed by unpaired two-tailed Student’s p = 0.0161) and a reduced DAI score (p = 0.0011). Colon shortening is one of the main characteristics of DSS-induced colitis (p < 0.0001). In addition, the histopathological analysis showed a consistent trend. Specifically, in the DSS group, obvious structural disorder, epithelial damage, mucosal ulceration, gland swelling, and inflammatory cell infiltration were observed in colon sections compared to the control and FUC groups (p = 0.0319). These results demonstrate that L-fucose effectively ameliorates intestinal injury in DSS-induced colitis mice. However, it had no significant effect on the body weight, colon length, or intestinal mucosal structure of normal mice.To study the therapeutic effect of L-fucose on UC, a DSS-induced chronic colitis mouse model was established A. As sho colitis D,E. The C groups F,G. Signp = 0.0275) (p = 0.0009). The protein and mRNA levels of IL-22 in the colon were observed to be increased after L-fucose administration in DSS-induced mice  C,D. Like 0.0275) G. In addced mice H–J. ThesTo explore how L-fucose regulates intestinal IL-22 secretion, we screened IL-22-related transcription factors via qRT-PCR analysis and Western blotting. Among the four important transcription factors, Notch, Hes and STAT5 showed no significant difference between the DSS and DSS + FUC groups A–C. Whil+ T cells, we further investigated the specific mechanism by which L-fucose mediated intestinal IL-22 secretion in in vitro experiments of L-fucose intervention in CD4+ T lymphocytes, in the presence and absence of an AHR inhibitor (CH223191). The results of flow cytometry showed that L-fucose significantly increased the number of IL-22 positive cells in CD4+ T cells (p = 0.0309), while CH223191 inhibited this function of L-fucose (p = 0.007) , while CH223191 suppressed the nuclear expression of AHR (p = 0.0196) (+ T cells (+ T cells in the L-fucose group (p = 0.0233) (+ T cells (p = 0.0299). The transcription level of CYP1A1 in CD4+ T cells among the three groups showed similar trends (+ T cells by upregulating the transcription factor AHR.Since IL-22 in the mouse gut is partly derived from CD4= 0.007) A,B. In aCH223191 C,D. The  0.0196) E,F. qRT- T cells G. Furthe 0.0233) H,I. CH22r trends J. Based + T cells, intestinal IL-22 is also largely derived from the innate immune cell ILC3 [+ROR-γt+ cells was significantly increased after L-fucose intervention (p = 0.0295). In contrast, CH223191 inhibited the IL-22-promotion effect of L-fucose (p = 0.0084). In addition, qRT-PCR analysis of IL-22 mRNA in LPMCs and ELISA assay of IL-22 in culture supernatant of LPMCs showed similar trends  that is mainly distributed on nonhematopoietic stem cells, and the IL-22/IL-22R axis plays a key pivotal role between immune function and intestinal barrier function connections . As showTo demonstrate whether L-fucose promotes ISC regeneration through IL-22, we induced an organoid injury model in the coculture system with TNF-α. ELISA showed that the TNF-α level was significantly increased in the medium of the coculture system after TNF-α induction, indicating the successful establishment of an inflammatory model I. As sho+ T cells in mouse spleen cells and ILC3 cells in LPMCs through activation of nuclear AHR. Further studies showed that the stimulatory effect of L-fucose on ISC regeneration and proliferation was mediated by IL-22 being released from LPMCs in the LPMC-intestinal organoid coculture experiment. These results indicated that L-fucose indirectly acted on ISCs through LPMCs, thus helping the recovery of damaged intestinal epithelium (Here, our study demonstrated that L-fucose could alleviate colon inflammation and restore the intestinal epithelial barrier in DSS-induced colitis mice, and it worked through promoting the regeneration and proliferation of ISCs. In addition, in the presence of L-fucose, IL-22 and its related transcription factor AHR were significantly elevated in the colons of colitis mice. In an in vitro experiment, we found that L-fucose stimulated the secretion of IL-22 from CD4ithelium .Inflammatory mucosal healing is the primary goal of IBD treatment to improve clinical symptoms, disease recurrence and resection-free survival. ISCs can generate various types of mature intestinal epithelial cells, so the maintenance of their stemness is the key to the continuous renewal and repair of the intestinal mucosal epithelium after injury ,26. As a+ T cells, acts as an important regulator of the DNA damage response in ISCs. Given the important role of IL-22 in the maintenance of ISC homeostasis, we found that the exogenous administration of L-fucose significantly increased intestinal IL-22 secretion and IL-22R expression in DSS-induced chronic colitis. The in vitro experiments also showed increased production of IL-22 from CD4+ T cells and ILC3 cells after L-fucose treatment.It is well known that the intestinal immune microenvironment, including innate and adaptive immunity, is related to the maintenance of intestinal homeostasis . With th−/− mice typically exhibit mild spontaneous enteritis [−/− mice than in wild-type mice [−/− mice are more susceptible to Citrobacter infection [+ T cells and LPMCs, we found that the mRNA levels of AHR and its target gene, CYP1A1, were significantly elevated in the colons of colitis mice by L-fucose treatment. The increased nuclear AHR protein in colon cells indicated the activation of AHR signaling. In an in vitro experiment, the AHR inhibitor CH223191 significantly reduced the stimulatory effect of L-fucose on IL-22 secretion from CD4+ T cells and LPMCs, which demonstrated that L-fucose promoted IL-22 release through the activation of AHR in CD4+ T cells and LPMCs. However, the examination of the role of AHR was solely based on pharmacological inhibition, and the absence of AHR gene deletion in CD4+ T cells and LPMCs was a limitation of this study.The literature has reported that AHR-mediated signaling controls the production of IL-22 . AHR is nteritis , DSS-indype mice , and AHRnfection . To explIL-22R is widely distributed in the cell membrane of ISCs and is used to receive IL-22 signaling . We founIn summary, our data demonstrated that L-fucose played a therapeutic role in ameliorating DSS-induced chronic colitis in mice by accelerating ISC proliferation, and it works through the AHR/IL-22 pathway of LPMCs. These results prove that L-fucose may be a promising dietetic treatment for IBD. However, further research is needed to verify the specific therapeutic effect and mechanism in clinical trials.


In the current study, we generated a CerS6 mouse using CRISPR-Cas9, in which the DDRSDIE motif was replaced by ADAAAIA. While levels of CerS6ADAAAIA expression were unaffected in the CerS6ADAAAIA mouse, and CerS6ADAAAIA was able to generate C16-ceramide in vitro, ceramide levels were significantly reduced in the CerS6ADAAAIA mouse, suggesting that replacing this motif affects an as-yet unknown mechanism of regulation of ceramide synthesis via the DDRSDIE motif in vivo. Crossing CerS6ADAAAIA mice with CerS5 null mice led to generation of viable mice in which C16-ceramide levels were reduced by up to 90%, suggesting that depletion of C16-ceramide levels is compensated for by other ceramide species with different acyl chain lengths.The important membrane lipid, ceramide, is generated by a family of homologous enzymes, the ceramide synthases (CerSs), multi-spanning membrane proteins located in the endoplasmic reticulum. Six CerS isoforms exist in mammals with each using a subset of acyl-CoAs for (dihydro)ceramide synthesis. A number of mice have been generated in which one or other CerS has been genetically manipulated, including complete knock-outs, with each displaying phenotypes concomitant with the expression levels of the CerS in question and the presumed biological function of the ceramide species that it generates. We recently described a short C-terminal motif in the CerS which is involved in CerS dimer formation; deleting this motif had no effect on the ability of the CerS to synthesize ceramide  For inscyl CoAs  whereas cyl CoAs , 10. Whi (d18:1) , 11.in vitro CerS assays, or via metabolic labeling using NBD-sphinganine (NBD-Sph), but did affect the ability of CerS to form homo- or heterodimers as determined by co-immunoprecipitation, leading to the suggestion that the DxRSDxE motif is involved in CerS dimer formation [The CerS are part of a larger gene family, the Tram-Lag-CLN8 (TLC) domain family , 13. We ormation .ADAAAIA mouse is able to synthesize ceramide in vitro at levels similar to the CerS6 wild type (WT) mouse. However, C16:0-ceramide and C16:0-dihydroceramide levels, measured by mass spectrometry, were 30–40% lower in the small intestine (jejunum) and in the liver compared to the CerS6 mouse, which is similar to the extent of C16-ceramide reduction in the CerS6 null mouse [ADAAAIA mouse was crossed with CerS5 null mice, C16:0-ceramide and C16:0-dihydroceramide levels were depleted by up to 90%, although the mice survived into adulthood. The molecular basis by which ceramide levels are depleted in the CerS6ADAAAIA mouse is unknown, but limits the usefulness of this mouse to determine the precise role that the DxRSDxE motif plays in CerS in vivo. However, the viability of the CerS6ADAAAIA/CerS5 null mice, despite the ~90% depletion of C16-ceramide levels, suggests it could be a useful tool to delineate the precise roles of C16-ceramide in vivo.To further explore the physiological role of the DxRSDxE motif, we have now generated, using CRISPR-Cas9, a mouse in which this motif in CerS6 (DDRSDIE) has been replaced by ADAAAIA. The CerS6ll mouse . MoreoveNBD-sphinganine and palmitoyl-CoA (C16-CoA) were from Avanti Polar Lipids . Defatted-bovine serum albumin, a protease inhibitor cocktail and an anti-CerS6 antibody were from Sigma-Aldrich . Horseradish peroxidase was from the Jackson Laboratory . An enhanced chemiluminescence (ECL) detection system was from Cyanagen . An anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody and silica gel 60 thin layer chromatography plates were from Merck . All solvents were of analytical grade and purchased from Bio-Labs .ADAAAIA mice by CRISPR-Cas9 [5′- AGCTAGATTCAATGTCACTC-3′. The guide sequence was chosen by optimizing the lowest off-target [5’-ACCCTGCTGGTCCTAGGAAGCTGGTGACACTCCCATCTTTGTATTCTGCAGGTATCCAAGGCTGACGCGGCTGCCATTGCATCTAGCTCAGATGATGAGGACTCAGAGCCTCCAGGGAAGAAACCACACTCTTCAACAACC-3’. Primers used for genomic screening were as follows: forward 5′-CATTATGTTCCTGCACCACC-3′; reverse 5′-ATTCACAGACAGGACACATG-3′, to yield a 702 base pair fragment that was cleaved by either BsaWI or BsrDI restriction endonucleases to identify the WT allele or edited alleles, respectively. Mouse sequences were analyzed using Sequencher v5.4  and visualized using the University of California, Santa Cruz Genome Browser [C57BL/6JOlaHsd mice were purchased from Envigo  and used to generate CerS6 SPR-Cas9 . The CRIf-target , 18 and f-target . The sin Browser .tm2b(KOMP)Mbp (CerS5 null) mice (stock #022819) were purchased from Jackson Laboratories . Primers for genotyping were as follows: CerS5, forward 5’-GATGGGAGTTTTTCTTTGTGC-3’; reverse, 5’- TGATGGATTGGGCCTAGTGT-3’; CerS5 null forward 5’- CGGTCGCTACCATTACCAGT-3’, reverse 5’-TTGCCTCAAAACCCACCTAC-3’. CerS6 ADAAAIA mice were mated with CerS5 null mice to generate CerS6 ADAAAIA/CerS5 null mice.CerS5Animals experiments were approved by the Weizmann Institute of Science Animal Care Committee. Mice were maintained under special pathogen-free conditions and treated according to the Animal Care Guidelines of the Weizmann Institute of Science Animal Care Committee and the National Institutes of Health’s Guidelines for Animal Care.2 containing a protease inhibitor cocktail (Sigma-Aldrich). Protein was determined using the BCA reagent (Cyanagen). Cell homogenates were incubated with 15 μM NBD-Sph, 20 μM defatted-bovine serum albumin and 50 μM C16-CoA in a 20 μl reaction volume at 37°C. Reactions were terminated by addition of chloroform/methanol  and lipids extracted [2 and resuspended in chloroform/methanol  and applied to thin layer chromatography plates . The thin layer chromatography plates were developed using chloroform/methanol/2M NH4OH . NBD-labelled lipids were visualized using an Amersham Typhoon 5 biomolecular fluorescence imager and quantified by ImageQuantTL .CerS assays were performed as described , 23. Mouxtracted . Lipids ADAAAIA group, which used 2 females and 2 males.Tissues were homogenized in double-distilled water containing protease inhibitors using a GentleMACS Dissociator . Ceramide and dihydroceramide levels were determined by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) using an ABI 4000 quadrupole-linear ion trap mass spectrometer , 25. GroWestern blotting was performed as described . Mouse tADAAAIA mice were generated using CRISPR-Cas9   , by ~60%in liver , similar to zero , with a  to zero . In CerSn length . While tΔ79–120 was confirmed to be catalytically active in vitro, although with a small reduction in activity [Δ79–120 was not expressed due to exon skipping in exons 3 and 4 [Δ84–118 mice, which also resulted in exon skipping, and lack of protein production (unpublished studies). We also generated CerS2 mice with the C-terminal DXRSD motif replaced by alanine residues, as well as mice lacking the C-terminus, but due to intron retention, CerS2 was not expressed.A number of mice have been generated in which CerS have been genetically modified, although most of them were generated so as to completely delete the CerS in question , 28–30. activity . Unlike  3 and 4 . Similarin vivo. Supporting this notion was the observation that the CerS6ADAAAIA mouse was able to generate C16-ceramide in vitro, although, unfortunately it was unable to generate ceramide in vivo. This is somewhat reminiscent of previous observations that levels of ceramide in cultured cells upon deletion, or manipulation of various functional sequences in CerS does not always correspond to direct enzymatic assays in vitro [in vitro and in vivo [in vitro, but that their in vivo function affects CerS activity in such a way that cannot be reproduced in vitro. For example, FATP2 which interacts with CerS2 in mouse liver, increases ceramide levels upon overexpression in Hek293T cells [in vivo [in vivo, at least in mouse models, which is likely to lead to further refinement of our understanding of their complex modes of regulation.Despite the inability in previous studies to generate CerS mice defective in one or other functional domain, we nevertheless felt that the DDRSDIE motif in CerS was an attractive option to attempt to test a functional role of a particular domain in vitro , 34. Thi in vivo , 36, and in vivo , 34. It 3T cells  and ELOV[in vivo . TogetheS1 Raw images(PDF)Click here for additional data file.


MET2 gene from Candida albicans encoding L-homoserine O-acetyltransferase (CaMet2p), an enzyme catalyzing the first step in that pathway, was cloned and expressed as the native or the oligo-His-tagged fusion protein in Escherichia coli. The recombinant enzymes were purified and characterized for their basic molecular properties and substrate specificities. The purified MET2 gene product revealed the appropriate activity, catalyzed the conversion of L-homoserine (L-Hom) to O-acetyl-L-homoserine (OALH), and exhibited differential sensitivity to several L-Hom or OALH analogues, including penicillamine. Surprisingly, both penicillamine enantiomers (L- and D-Pen) displayed comparable inhibitory effects. The results of the docking of L- and D-Pen to the model of CaMet2p confirmed that both enantiomeric forms of the inhibitor are able to bind to the catalytic site of the enzyme with similar affinities and a similar binding mode. The sensitivity of some fungal cells to L-Pen, depending on the presence or absence of L-Met in the medium, clearly indicate Met2p targeting. Moreover, C. glabrata clinical strains that are resistant to fluconazole displayed a similar susceptibility to L-Pen as the wild-type strains. Our results prove the potential usefulness of Met2p as a molecular target for antifungal chemotherapy.Due to the apparent similarity of fungal and mammalian metabolic pathways, the number of established antifungal targets is low, and the identification of novel ones is highly desirable. The results of our studies, presented in this work, indicate that the fungal biosynthetic pathway of L-methionine, an amino acid essential for humans, seems to be an attractive perspective. The  Candida, especially C. albicans and C. glabrata, the filamentous fungi of Aspergillus spp., and Cryptococcus spp. [C. albicans is considered as the fourth most popular etiological agent of nosocomial infections worldwide [Mycoses are caused by various fungal species; however, life-threatening systemic fungal diseases are mainly entailed by human pathogenic yeasts belonging to the genera cus spp. . C. albiorldwide . In receorldwide . Moreoveorldwide . The curC. albicans genes identified as essential and not having counterparts in mammalian systems [Only very few of 130  systems  have bee systems ,7. The d systems , well beO-acetyltransferase EC 2.3.1.31 (Met2p), converting L-homoserine (L-Hom) to O-acetyl-L-homoserine (OALH).L-methionine is synthesized in fungal cells through the direct sulfhydrylation pathway or the transsulfurylation pathway, from L-homoserine or L-cysteine, respectively . The firCryptococcus neoformans in a mouse inhalation model [MET2 gene in this fungus caused methionine auxotrophy that could be rescued via the addition of >60 µM L-methionine to the growth medium [Candida albicans, Candida guilliermondii, and Saccharomyces cerevisiae mutant cells depleted in MET2 genes turned out to be methionine auxotrophs [O-acetyltransferase has therefore been proposed as one of the potential molecular targets for antifungal drugs [C. neoformans [Met2p is undoubtedly a potential molecular target for novel antifungal chemotherapeutics. Nazi et al. discovered that this enzyme is essential for the virulence of on model . The dish medium . This coh medium . Additioxotrophs ,11,12. Lal drugs . Neverthoformans , possiblO-acetyltransferase is also essential in bacterial cells. Mycobacterium tuberculosis cells lacking the META gene, encoding the bacterial homolog of Met2p, were unable to induce infection in an vivo mouse inhalation model or proliferate inside human macrophages, and they could not grow in L-methionine-free medium [It is worth mentioning that L-homoserine e medium . Chaton e medium .O-acetyltransferase as a molecular target in antifungal chemotherapy. The MET2 gene from C. albicans was cloned and expressed as the native or the oligo-His-tagged fusion protein in E. coli. The recombinant enzymes were purified and characterized for their basic molecular properties and substrate specificities, and the enzyme inhibitory potentials of several L-homoserine or L-methionine structural analogs were evaluated.In this paper, we present the results of our studies on the determination of the potential usefulness of L-homoserine MET2 gene , encoding putative L-homoserine O-acetyltransferase from C. albicans (CaMet2p), was retrieved from the Candida Genome Database [CaMet2p amino acid sequence and the sequences encoding L-homoserine O-acetyltransferases; with defined crystal structures from Haemophilus influenzae (34%) (PDBID: 2B61), Staphylococcus aureus (28%) (PDBID: 4QLO), Mycobacterium smegmatis (32%) (PDBID: 6IOG), and putative Met2p from Saccharomyces cerevisiae (53%) (Saccharomyces Genome Database: YNL277W); and the presence of conserved and important residues.The Database . The expCaMet2p (≥50.34% identity) from 59 microorganisms enabled us to perform a multiple sequence alignment , Haemophilus influenza (PDBID: 2B61), and Leptospira interrogans (PDBID: 2PL5), respectively  was used as a template to prepare the AlphaFold model of the C. albicans enzyme [O-acetyltransferase from C. albicans and M. smegmatis showed that despite the barely 32% identity of sequences, the binding sites of these two enzymes are very well conserved   , as welles group . Notableequences .Ascomycota , and Arg225. All of these residues  are conserved in the Ascomycetes group , N-terminus His-tagged L-homoserine O-acetyltransferase (CaMet2NHp), and C-terminus His-tagged L-homoserine O-acetyltransferase (CaMet2CHp), were cloned into the pET101/D-TOPO expression vector, yielding the pET101/D-TOPO + MET2, pET101/D-TOPO + MET2NH, and pET101/D-TOPO + MET2CH plasmids. Plasmids containing the CaMET2 gene with an oligo-Histidine tag added to either the C- or N-terminus, were constructed to facilitate further purification of the protein. Additionally, a mutagenesis procedure was conducted because the CaMET2 gene contains a single CTG codon at position 490–492, which is translated as L-serine in C. albicans, but as L-leucine by E. coli cells. The obtained plasmids were sequenced and then E. coli BL21 Star (DE3) and E. coli Rosetta (DE3) pLysS competent cells were transformed with pET101/D-TOPO + MET2 and pET101/D-TOPO + MET2NH, respectively. The transformed cells were used for the overexpression of the cloned genes in a IPTG-inducible Tabor-Studier system, which allowed for the overproduction of CaMet2p, CaMet2CHp, and CaMet2NHp. Proteins were purified using the AKTA Fast protein liquid chromatography (FPLC) system. HisTrap™ Fast Flow metal ion affinity chromatography was used for CaMet2NHp purification. The target protein was eluted with 250 mM imidazole, with an 86% purity rate. CaMet2p was purified on an ion exchange Resource™ Q column and eluted with 16% NaCl, with a 44% purity rate  as substrates to produce OALH. Specific activities of the wild-type and N-terminus His-tagged enzyme were at a similar level, whereas they were significantly lower in the case of the C-terminus His-tagged enzyme version . The stability of CaMet2NHp and CaMet2p could be improved by adding glycerol up to 20% v/v. The enzyme preparation remained active for a longer period of time when kept at 4 °C , as well as the structure of the AlphaFold model  are located near the middle of the binding pocket, while the catalytic triad is located deep inside, at the bottom of the binding cavity. The part of the L-homoserine binding site, between these two regions, is formed by the Leu66, Ala65, Phe392, Tyr233, and Leu393 residues with their sidechains shaping a hydrophobic ring around this area (Comparable enzyme inhibitory properties of both penicillamine enantiomers (L- and D-Pen) are quite surprising, in the light of the expected stereospecificity of Met2p. Thus, we decided to perform the molecular docking analysis to check whether both isomers could bind to the active center. Since there is no X-ray structure of ld model  of CaMetive site  showed this area .CaMet2p confirmed that both enantiomers are able to bind to the binding site of the enzyme with similar affinities and similar binding modes. There is enough room in the active site to accommodate the changed pose of the ligand, resulting from its different Cα configuration, while the carboxy and alpha-amino moieties are still able to recreate the network of interactions of the natural substrate with the Asp390, Arg226, and Ser67 residues. The binding of the remaining, nonpolar part of the ligands is dominated by nonspecific van der Waals interactions with the surrounding hydrophobic ring, resulting in similar affinities of L- and D-penicillamine.The results of the docking of L- and D-Pen  to the mPenicillamine is a sulfur-containing, non-proteinogenic amino acid that was reported to inhibit several pyridoxal-5′-phosphate (PLP)-dependent enzymes . Both L-Candida species and S. cerevisiae, were determined by the serial two-fold dilution method in 96-well microtiter plate format. Minimal inhibitory concentrations (MICs) were determined in minimal YNB medium containing ammonium sulfate (SA) as a nitrogen source, supplemented with 10 mM L-methionine (L-Met) when indicated, and RPMI-1640 medium, as recommended by CLSI in the M27-A3 procedure. The results are summarized in The antifungal in vitro activities of the Met2p inhibitors, L- and D-Pen, against several C. albicans was previously studied in combination with fluconazole [C. albicans strains. The combined treatment increased the survival rate of G. mellonella larvae infected with C. albicans, as well as decreasing the fungal burden. Mechanism studies elucidated that the synergism is related to inhibition of the morphological transformation, the disruption of intracellular calcium homeostasis, and the activation of metacaspase, which is closely related to cell apoptosis. Our results also indicate that CaMet2p inhibition could be related to that synergistic effect. The D-penicillamine antifungal effect against conazole . The autC. glabrata clinical isolates to L-Pen. Six out of 8 examined strains were characterized as being resistant to fluconazole. All of the tested C. glabrata clinical isolates displayed similar susceptibilities to L-Pen as the wild-type strain in the YNB-SA medium , which involves the condensation of sulfide with O-acetyl-L-serine. The alternative direct sulfhydrylation pathway condenses sulfide with O-acetyl-L-homoserine, yielding L-homocysteine, which is then converted to L-cystathionine and next to L-cysteine via the reverse transulfuration pathway. Filamentous fungi such as Aspergillus nidulans and Neurospora crassa, as well as many yeasts such as C. albicans, can synthesize L-cysteine using both ways, whereas S. cerevisiae and C. glabrata lack the O-acetyl-L-serine pathway [C. glabrata is more closely related to S. cerevisiae than to C. albicans, as indicated from the phylogenetic analysis .Our results showed that L-Pen influenced the growth of B medium . The grothionine . S. cereof L-Pen . MoreoveC. albicans cells possessing both the pathways mentioned above [Interestingly, ed above , are senIn contrast to L-Pen, its D stereoisomer exhibited no antifungal activity in the YNB medium, regardless of L-Met supplementation . HoweverC. glabrata cells treated with L-Pen in YNB medium supplemented with ammonium sulfate -L-cysteine hydrochloride, and L-2-aminoadipic acid.All reagents were commercially available and purchased from Sigma-Aldrich, St. Louis, MO, USA. Reagents used for the determination of enzyme activity: AcCoA lithium salt, L-homoserine, guanidinium-HCl, and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB). Tested enzyme inhibitors included: acetyl-L-carnitine, D-penicillamine, L-penicillamine, 3-(2-thienyl)-L-alanine, L-methionine sulfoximine, DL-phosphinothricin, L-2,4-diaminobutyric acid dihydrochloride, DL-2-allylglycine, L-canavanine, Escherichia coli One ShotTM TOP10 cells  were used in the cloning procedures. Escherichia coli One ShotTM BL21 StarTM (DE3) and Escherichia coli Rosetta (DE3) pLysS competent cells were used to produce wild-type and recombinant proteins, respectively.Candida albicans ATCC 10231, Candida parapsilosis ATCC 22019, Candida krusei ATCC 6258, Candida glabrata ATCC 90030, Candida famata DSM 3428, Candida rugosa DSM 2031, Candida dublinensis CBS 7987, and Saccharomyces cerevisiae ATCC 9763; and 8 C. glabrata clinical isolates: CZD 6, CZD 209, GD 211, GD 310, CZD 342, CZD 373, CZD 377, and CZD 513. Clinical strains were isolated from patients from the Children’s Memorial Health Institute in Warsaw (CZD), or the Medical University of Gdansk (GD), in 2011 and 2012 [Antimicrobial activity of penicillamine was tested on 8 reference strains: and 2012 . −1) or chloramphenicol (34 µg·mL−1) when required), and incubated at 37 °C for 6–24 h.All of the bacterial strains used were cultured on solid (with 1.5% (m/V) agar) and in liquid Luria-Bertani (LB) media  and grown at 30 °C for 18 h.MET2 gene was amplified via polymerase chain reaction (PCR) using the Candida albicans SC5314 genome isolated with Genomic Mini AX YEAST kit  as a matrix, and Phusion High-Fidelity PCR Master Mix with HF Buffer . Primers used for the amplification of the wild-type Met2p gene were 5′-CACCATGACATACAAAGACGTGACA-3′ as a forward primer and 5′-GATTATTCAATTATTCAAAAAACTAGTGATGAAAC-3′ as a reverse primer. Primers used for the amplification of the oligoHis-tagged Met2NHp gene were 5′-CACCATGCATCATCATCATCATCATACATACAAAGACG-3′ as a forward and 5′-TTGCATCCCTTGATTATTCAATTATTCAAAAAACTAGTGATG-3′ as a reverse primer; primers used for amplification of the oligoHis-tagged Met2CHp gene were 5′-CACCATGACATACAAAGACGTGACA-3′ as a forward and 5′-TCAATGATGATGATGATGATGATTATTCAAAAAACTAGT-3′ as a reverse primer; introduced poly-Histidine domains are marked in bold. The PCR temperature gradient profile was: initial denaturation at 98 °C for 10 s, 30 cycles of denaturation at 98 °C for 1 s, starter annealing at 58 °C for MET2 and 68 °C for MET2NH for 5 s, and elongation at 72 °C for 30 s, followed by elongation at 72 °C for 60 s. The formed PCR products  were cloned into the pET101/D-TOPO vector using the Champion™ pET101 Directional TOPO™ Expression Kit (Invitrogen), and transformed into E. coli One ShotTM TOP10 cells . Plasmids were isolated with the use of the Plasmid Mini AX kit , and subjected to mutagenesis procedure. The constructed plasmids were sequenced in the coding region and transformed into E. coli One ShotTM BL21 StarTM (DE3) and E. coli Rosetta (DE3) pLysS competent expression cells in the case of the wild-type and His-tagged protein, respectively. The MET2 or pET101/D-TOPO +MET2NH DNA matrix. The reaction mixture was subjected to PCR with a temperature gradient profile of an initial denaturation at 98 °C for 10 s, 30 cycles of denaturation at 98 °C for 1 s, starter annealing at 58 °C for 5 s, and elongation at 72 °C for 30 s, followed by elongation at 72 °C for 60 s. The obtained PCR products were digested with Fast Digest DpnI restriction enzyme  in 37 °C for 30 min, and subjected to gel electrophoresis, after which undigested (7000 bp) fragments were cut out from the agarose gel and the DNA was isolated according to the Gen Elute™ Gel Extraction Kit protocol . Isolated DNA was suspended in 50 µL water and used for a ligation procedure using T4 DNA Ligase Buffer (10×)  and T4 DNA Ligase (5 U·µL−1) . The ligation reaction was performed for 30 min in 22 °C, and the obtained plasmids containing mutated MET2 and MET2NH genes were used for the transformation of E. coli One ShotTM TOP10  competent cells.The mutagenesis reaction mixture contained Phusion High-Fidelity PCR Master Mix with HF Buffer , and forward 5′P-CATTCCCCCCATGGATCCTCCA-3′ and reverse 5′P-GCATTAGAATACTCGGCAATTTACAACAATA-3′ primers, and pET101/D-TOPO +Proteins separated in 10% polyacrylamide gel were subjected to electroblotting to a nitrocellulose membrane for 1 h at 20 mA. A nitrocellulose membrane with transferred proteins was then placed in 5% skim milk wash buffer solution  and left overnight. The membrane was washed 3 times with 10% (by volume) wash buffer, and next incubated for 1 h in antibody solution  BSA, 0.05% (by volume) Tween20, anti-His6 0.033% (by volume) antibodies )). The detection of proteins conjugated to antibodies was made with 1 mL of 3,3′,5,5′-tetramethylbenzidine (TMB) liquid substrate system for membranes .MET2 and pET101/D-TOPO + MET2NH were transformed into E. coli One ShotTM BL21 StarTM (DE3) and E. coli Rosetta (DE3) pLysS competent cells, respectively. Expression cells were grown overnight at 37 °C in LB medium supplemented with ampicillin and chloramphenicol in the case of E. coli Rosetta (DE3) pLysS cells. A total of 10 mL of starter culture was added to 800 mL of LB medium supplemented with ampicillin and chloramphenicol, and incubated at 37 °C up to an optical density (OD600) = 1.0. Induction was made with isopropyl-β-d-thiogalactoside (IPTG) to a final concentration of 1 mM. Cells were cultured for 6 h at 37 °C. Cells were harvested via centrifugation for 20 min at 4000 rpm in 4 °C. The obtained cell pellets were stored for later use at −20 °C.The constructed plasmids pET101/D-TOPO + 2, and centrifuged for 20 min at 12,000 rpm in 4 °C. To dispose of the excess salt in the mixture, 20% polyethylene glycol 6000  was added and centrifuged for 20 min at 12,000 rpm in 4 °C. The cell pellet was suspended in 10 mL buffer 1 supplemented with cOmplete™ Protease Inhibitor Cocktail , 10 mM EDTA, and 10 mM DTT. The solution was centrifuged and loaded onto the Resource™ Q column . The elution of the proteins was conducted in a rising concentration of NaCl up to 0.5 M concentration in elution buffer 2 (20 mM phosphate buffer of pH 7.0 and 1 M NaCl) in 10 column volumes.The bacterial pellet was suspended in buffer 1 (20 mM sodium phosphate buffer pH 7.0) supplemented with cOmplete™ Protease Inhibitor Cocktail , 10 mM EDTA, and 10 mM dithiothreitol (DTT). The mixture was sonicated on ice  and centrifuged for 20 min at 10,000 rpm. Streptomycin sulphate was added the supernatant to a final concentration of 1.2%, and the mixture was centrifuged for 20 min at 12,000 rpm at 4 °C. The obtained supernatant was saturated with 36.2% ammonium sulphate, mixed in an ice bath for 20 min, and then centrifuged for 20 min at 12,000 rpm at 4 °C. During this step, the protein mixture was initially purified, and the precipitate containing the examined proteins was dissolved in buffer 1 containing 40 mM NaCl and 10 mM MgCl5  supplemented with cOmplete™ Protease Inhibitor Cocktail  and 10 mM DTT. The mixture was sonicated on ice  and centrifuged for 20 min at 10,000 rpm. A total of 10 mL of supernatant was loaded onto a 5 mL HisTrap™ Fast Flow  column equilibrated with binding buffer. Elution was performed in a linear rising imidazole concentration. Elution buffer W500: 20 mM sodium phosphate buffer pH 8.0, 500 mM imidazole, 500 mM NaCl, and 1 mM Tween 20 ). The bacterial pellet was suspended in binding buffer WDensitometric analysis measuring the purity rate of the purified protein was performed with the Gel analyzer program .The assay was conducted according to the method of Foyn et al. . A totalThe kinetic parameters were determined for both the wild-type and the His-tagged version of the enzyme by measuring the activity of 300 nM protein according to the activity assay procedure. The reaction mixture contained various concentrations of substates: 0–4 mM AcCoA and 0–6 mM L-Hom at a constant concentration of other substrates, 10 mM L-Hom and 3.5 mM AcCoA, respectively. 4 (pH 6.0–8.0), and glycine (pH 8.5–11.0). The determination of the optimal reaction buffer was made by comparing the activity of the enzyme in the above reaction buffers at pH 8.0–8.5. The optimal pH for the enzyme activity was determined according to the activity assay procedure with the changing pH of the buffers used: Tris-HCl (pH 8.5–9.0), HEPES (pH 7.0–8.0), NaHPOCaMet2NHp. The influence of the inhibitor on the activity of the enzyme was determined by the measurement of the difference in the absorbance at 412 nm, assessed according to the above method. For the assessment of the inhibitory assay, 1–15 mM of inhibitor was added to the activity reaction buffer. The reaction was commenced via the addition of 300 nM of 4 colony-forming units (CFU)/mL in a Yeast Nitrogen Base (YNB) without amino acids and ammonium sulfate , supplemented with or without 0.05–10 mM L-methionine and Roswell Park Memorial Institute medium 1640 (RPMI) . Plates were incubated at 37 °C for 24 h; the rate of growth was determined by measuring the optical density at 600 nm using a microplate reader . The MIC50 and MIC90 parameters were defined as the lowest concentration of antifungal compound that inhibited fungal growth by at least 50% or 90%, respectively. Minimum inhibitory concentrations (MICs) were performed using the modified M27-A3 procedure specified by the CLSI . In 96-wM. smegmatis (PDBID: 6IOH) was used as a template to prepare the AlphaFold model of the C. albicans enzyme [C. albicans enzyme were used as the receptors for the docking analysis. The structures of all ligands were built using the HyperChem software [The structure of the enzyme from s enzyme . The crysoftware . All docsoftware ,52 afterThe center of the docking grid was set to the geometric center of the bound ligand and its size was set to 40 Å in each direction, to cover not only the binding site, but also the majority of the protein subunit. The adequacy of the docking protocol, as well as the forcefield used, was verified by redocking of the rebuilt L-HOM, as mentioned earlier. The geometry of the native complex was restored, with the average RMS of the lowest energy cluster below 1.5 Å. Figures showing the results of molecular modeling were prepared using the VMD program .MET2 gene of C. albicans was unequivocally identified as coding for L-homoserine O-acetyltransferase, catalyzing the first step in the L-Met biosynthesis pathway. The CaMET2 was cloned and expressed in E. coli as native and His-tagged fusion proteins. The recombinant CaMet2p was only slightly inhibited by the end pathway product, i.e., L-Met, at a non-physiological concentration; thus, the mechanism of feedback inhibition seems not to play any role in amino acid biosynthetic pathway regulation. The L- and D-penicillamine inhibitory potential against CaMet2NHp, as well as both compounds’ antifungal activities, prove the usefulness of Met2p as a molecular target for antifungal chemotherapy. For this reason, the development of inhibitors with a stronger activity than this displayed by L-Pen and D-Pen is worth trying.In conclusion, the


Site directed mutagenesis was used to generate additional variants. VHH fusions with neurotensin (NT) allowed rapid, hypothermia-based screening for VHH-mediated BBB transcytosis in wild-type mice. One anti-mTfR VHH variant was fused with an anti-amyloid-beta (Aβ) VHH dimer and labeled with fluorescent dye for direct assessment of in vivo target engagement in a mouse model of AD-related Aβ plaque pathology. An anti-mTfR VHH called M1 and variants had binding affinities to mTfR of <1nM to 1.52nM. The affinity of the VHH binding to mTfR correlated with the efficiency of the VHH-NT induced hypothermia effects after intravenous injection of 600 nmol/kg body weight, ranging from undetectable for nonbinding mutants to -6°C for the best mutants. The anti-mTfR VHH variant M1P96H with the strongest hypothermia effect was fused to the anti-Aβ VHH dimer and labeled with Alexa647; the dye-labeled VHH fusion construct still bound both mTfR and Aβ plaques at concentrations as low as 0.22 nM. However, after intravenous injection at 600 nmol/kg body weight into APP/PS1 transgenic mice, there was no detectible labeling of plaques above control levels. Thus, NT-induced hypothermia did not correlate with direct target engagement in cortex, likely because the concentration required for NT-induced hypothermia was lower than the concentration required to produce in situ labeling. These findings reveal an important dissociation between NT-induced hypothermia, presumably mediated by hypothalamus, and direct engagement with Aβ-plaques in cortex. Additional methods to assess anti-mTfR VHH BBB transcytosis will need to be developed for anti-mTfR VHH screening and the development of novel MRI molecular contrast agents.The blood-brain barrier (BBB) presents a major obstacle in developing specific diagnostic imaging agents for many neurological disorders. In this study we aimed to generate single domain anti-mouse transferrin receptor antibodies (anti-mTfR VHHs) to mediate BBB transcytosis as components of novel MRI molecular contrast imaging agents. Anti-mTfR VHHs were produced by immunizing a llama with mTfR, generation of a VHH phage display library, immunopanning, and  Alzheimer’s disease (AD) is one of the most important causes of dementia in the elderly . About 6The blood-brain barrier (BBB) represents a significant obstacle in delivering diagnostic and therapeutic agents to the central nervous system (CNS), preventing uptake of more than 98% of potential neurotherapeutics to brain –14. The BBB crossing based on receptor mediated transcytosis (RMT) is potentially especially promising. The use of protein shuttles has the potential to facilitate the transport of therapeutic agents across the BBB using specific endogenous receptor systems. Candidate receptor systems including transferrin receptors (TfR), low-density lipoprotein receptors, insulin receptors and neuropeptide receptors are highly expressed on the BBB where they mediate RMT . Among tCamelids produce functional antibodies devoid of light chains called heavy chain-only antibodies (HCAbs) , 27. HCAin vivo would be helpful since in vitro models may not be fully predictive of in vivo results [Current methods (ELISA or radioisotope detection) to evaluate BBB crossing require substantial resources and can be time-consuming , 47, 48. results . Neurote results . NT is e results . It was  results , 53. The results . Injecti results . Meanwhi results . On the  results , 47. TheFig 1). We hypothesized that this modular screening system can be used to identify and optimize anti-mTfR VHHs for BBB transcytosis and brain target binding.Here, we independently generated additional mTfR-binding VHHs and used a similar NT based modular system to screen these VHHs for mTfR-mediated BBB transcytosis  was immunized under contract agreement through Triple J Farms  following the method previously described [A single adult male llama (escribed . Briefly5′-GTCCTGGCTGCTCTTCTACAAGG-3′ and CALL002: 5′-GGTACGTGCTGTTGAACTGTTCC-3’). The heavy-chain specific amplicon was isolated using electrophoresis with low-melting point agarose extraction with the QIAquick Gel Extraction kit (Qiagen). A secondary amplification was performed using a modification of the primers (VHH-Esp-For: 5′-CCGGCCATGGCTGATGTGCAGCTGCAGGAGTCTGGRGGAGG-3′ and VHH-Esp-Rev: 5′-GTGCGGCCGCTGAGGAGACGGTGACCTGGG T-3′) used by Pardon et al. to facilitate cloning into the phagemid pHEN2 [7 independent clones. Phage was produced for screening using the M13KO7 helper phage  followed by precipitation by addition of one-fifth volume 20% polyethylene glycol 6000 / 2.5 M sodium chloride solution on ice and centrifugation to purify the phage particles.The generation of an immune phage display library and isolation of mTfR binding VHH clones was performed using the methods previously described . Brieflyid pHEN2 . The amp11 phage) was mixed with blocking solution to a final volume of 500 μL and then transferred into the RIA tube to allow for association at room temperature and 600 rpm mixing. The RIA tube was then washed 20 times with 1x PBS and then the bound phage eluted with 100 mM triethylamine solution for 20 mins. The eluted phage solution was neutralized with 1:10 volume 1 M Tris-HCl, pH 8.0. The eluted phage was amplified in TG-1 cells and a second round of immunopanning was performed.Selection of mTfR specific VHH was performed using direct binding of phage to immobilize mTfR. Standard radioimmunoassay (RIA) tubes were coated with 500 μL mTfR solution at 5 μg/mL in sodium carbonate buffer, pH 9.6 overnight at 4°C. The coating solution was removed, and the RIA tube filled with a 2% (w/v) non-specific blocking solution  in 1x Phosphate Buffered Saline (PBS). Amplified phage  to a final concentration of 1 mM and incubation overnight at 37°C. The culture blocks were centrifuged to pellet the cells and frozen at -80°C for 1 hour following removal of the culture supernatant. The culture block was then equilibrated to room temperature and 500 μL 1xPBS added to each well followed by shaking at 1500 rpm to resuspend the cell pellets and allow for release of VHH from the cells. The culture block was centrifuged for 20 min at 2000xg. Nunc Maxisorp plates were coated with mTfR at 1 μg/mL as described above and blocked with 1% bovine serum albumin (BSA). The clarified VHH supernatants were incubated on the mTfR plates for 1 hour at room temperature. The assay plate was washed and peroxidase conjugated goat anti-alpaca VHH domain specific antibody  at 0.8 μg/mL was transferred to the plate and incubated for 1 hour at room temperature. Following a final wash, the assay was developed by addition of tetramethylbenzidine  and absorbance was measured at 650 nm on a Biotek Synergy 2 plate reader. Clones with absorbance values greater than two standard deviations above background were considered of interest and subsequently sequenced.E. coli. Additional sequence, including a (Gly-Gly-Gly-Ser)3 between VHH domains, was incorporated for cloning into pHEN2 as a tandem dimer as part of the heterotrimer clones synthesized with TfR binders. To reduce the potential for recombination events, the DNA sequence was manually curated to adjust the codon usage and reduce the frequency of repetitive sequence within the Nb3-Nb3 dimer. The affinity of the Nb3-Nb3 dimer was measured using bio-layer interferometry as described below.Paraschiv et al. previously reported the isolation of amyloid beta binding VHH clones . We seleThe binding kinetics of the selected VHH clone against mTfR and Aβ was assessed by BLI. For measurements of mTfR kinetics, biotinylated VHH was diluted into assay buffer at 1 μg/mL and immobilized onto streptavidin coated biosensors  to a minimum response value of 1 nm on the Octet Red96 System (Sartorius). For measurements of amyloid beta, beta-amyloid(1–40)-Lys(biotin-LC) , was diluted into assay buffer at 1 μg/mL and immobilized onto streptavidin coated biosensors  to a minimum response value of 1 nm on the Octet Red96 System (Sartorius). Purified mTfR or VHH clones were diluted into assay buffer at the specified concentrations. The immobilized antigen biosensors were allowed to associate at 37C° followed by dissociation in the baseline buffer well location. All assays included a background correction condition to allow for sensor normalization. The ForteBio Data Analysis suite was used to normalize the association curves following background subtraction and Savitzky-Golay filtering. Curve fitting was applied using global fitting of the sensor data and a steady state analysis calculated to determine the association and dissociation constants. All assay steps were prepared in Greiner 96-well plates (#655209) in a volume of 300 μL. Assay buffer was defined as 0.1% BSA (w/v) in 1xPBS.Immunopanning and clone screening and BLI assessment of VHH binding kinetics sections, we synthesized several neurotensin-fused VHH monomers with different binding affinities to mouse transferrin receptor. In addition, we produced single polypeptide VHH heterotrimers that consisted of M1P96H, a (Gly-Gly-Gly-Ser)3 linker, Nb3, a (Gly-Gly-Gly-Ser)3 linker, and Nb3 using the method described in Amyloid beta specific VHH production. The VHH dimer Nb3-Nb3 binds to amyloid plaques in brain parenchymal of APP/PS1 mice. These constructs were termed “M1WT-NT”, “M1P96H-NT”, “M1AA-NT”, “M1R100dH-NT” and “M1P96H-triplet”. The VHH naming convention for P96H and R100dH are based on the Kabat nomenclature and refer to the specific amino acid residue modification positions within the VHH sequence [E. coli cells . Transformed cells were grown in terrific broth medium containing carbenicillin at 37°C and 300rpm shaking in baffled flasks. Once the culture density reached an optical density equal to 0.6, IPTG was added to a final concentration of 1mM to induce protein expression. For monomer VHH expression the post-induction incubation temperature was maintained at 37C but reduced to 30C for the M1P96H-triplet. Following overnight expression, cells were pelleted by centrifugation and VHHs were extracted through osmotic shock and recovery of the periplasmic fraction [Based on the methods described in sequence . Anotherfraction , 60. ClaP96H-triplets and H1-triplets were fluorescently labeled with using Alexa FluorTM 647 succinimidyl ester dye to allow fluorescence confocal microscopy of tissue sections. The triplets were incubated with the succinimidyl ester dye for 1 hour in 50mM sodium carbonate buffer, pH 9.6 and purified by desalting using a 5mL HiTrap desalting column in-line with the AKTA Pure. The binding fidelity of the VHH heterotriplet was assessed by ELISA against mTfR. The fluorescence dye labeled M1P96H-triplet and H1-triplet were named “M1P96H-triplet-647” and “H1-triplet-647”.M1Removal of contaminating endotoxin was achieved using High-Capacity Endotoxin Removal Resin  from the VHH preparations. A volume of 0.25ml endotoxin removal resin was added to 1ml of VHH sample. The VHH and resin were mixed for 2hr at room temperature to allow for absorption of endotoxin. The mixture was centrifuged to pellet the resin and the supernatant collected. A volume of 0.25ml fresh endotoxin removal resin was added to the first-pass solution and mixed for another 2hr at room temperature. Following a final centrifugation to pellet the resin, the solution was collected for endotoxin level testing. Endotoxin levels were measured using the Chromagenic Endotoxin Quant kit  according to the manufacturers protocol to ensure a level of endotoxin <0.5 endotoxin units/mg of total protein.To determine the effect on histidine mutation introduction, an ELISA was performed with the post VHH binding wash buffer at normal and reduced pH. Nunc Maxisorp plates were coated with mTfR at 1 μg/mL in 50mM sodium carbonate, pH 9.6 overnight at 4°C. The plates were blocked with 1% BSA in 1xPBS buffer and then triplicate dilutions of each VHH prepared in 0.5% BSA in 1xPBS buffer and transferred to the plate. Binding at room temperature for 2 hours was followed by three cycles of 5-minute washes with either 1xPBS, pH 7.2 or 1xPBS, pH 5.5. Following the pH dependent wash step, the bound VHH was detected with peroxidase conjugated goat anti-alpaca VHH domain specific antibody  at 0.8 μg/mL incubated for 1 hour at room temperature. Following a final wash, the assay was developed by addition of tetramethylbenzidine , the reaction was terminated by addition of 50 μL 1M hydrochloric acid and absorbance was measured at 450 nm on a Biotek Synergy 2 plate reader.WT-NT, M1P96H-NT, M1AA-NT, M1R100dH-NT and H1-NT for screening. Experiments were performed at the same time each day. To confirm the result from the five VHH-NT fusions, a blinded replication experiment was performed with additional fifteen mice randomized using a random number generator into five groups with three mice in each group. Six mice were randomly assigned into two groups to test the ability of VHH triplet-NT to get across the BBB after fused to anti-Aβ dimer Nb3-Nb3. Three mice were injected with M1P96H-Triplet-NT and three mice were injected with H1-Triplet-NT.All animal experiments were conducted under protocol approved by the National Institute of Neurological Disorders and Stroke (NINDS)/ National Institute on Deafness and Other Communication Disorders (NIDCD) Animal Care and Use Committee in the National Institutes of Health (NIH) Clinical Center (Protocol Number: 1406–21). C57BL/6J female mice were purchased from Jackson labs at 6–7 weeks of age and used at 7–12 weeks of age. Fifteen mice were divided into five groups with three mice in each group to test the five VHH-NT fusions. Anesthetized mice were injected via tail vein with VHH-NT fusions: M1SWE/PSEN1dE9 (MMRRC Strain #034832) positive transgenic mice were purchased from Jackson labs [P96H-triplet-647 and five mice were injected with H1-triplet-647.APPson labs , 62, maiWT-NT, M1P96H-NT, M1AA-NT, M1R100dH-NT and H1-NT were injected into wild-type C57BL/6J mice through the tail vein using a 30 Gauge needle in a single bolus at a dose of 600nmol/kg body weight in 1xPBS. The VHH-triplet-647s, M1P96H-triplet-647 and H1-triplet-647, were injected intravenously through single bolus injection at a dose of 1000nmol/kg body weight. Mice were maintained under anesthesia for approximately 2–3 minutes. Following the procedure, the mice were allowed to recover on a heating pad until fully ambulatory and then returned to their home cage with immediate access to food and water.Mice were anesthetized with 60% oxygen/ 40% medical air gas mixture containing 5% isoflurane in an induction box. After a stable anesthesia plane was established, mice were maintained at 1.5–2% isoflurane level. Artificial tears ointment was applied to prevent eye injury due to drying. Mice were placed on an electrical heating pad to maintain body temperature at 37C°. The VHH-NTs, M1Infrared thermometry was used for temperature measurement using an infrared thermometer . Abdominal fur was removed by application of topical depilatory cream prior to temperature measurement for precise data collection. Baseline temperature was measured three times before intravenous (IV) injection with a time interval of 30min between measurements. Then, mouse temperatures were measured at the time intervals of 30min, 1hr, 1.5hr, 2hr, 2.5hr, 3hr, 3.5hr, 4hr, 4.5hr and 5hr after injection. All temperature measurements were performed by investigators blinded to the identity of the injected VHH sample. Mice were briefly anesthetized (<20 seconds) with isoflurane for each temperature measurement. In preliminary experiments we confirmed that this brief anesthesia did not affect temperature.Two hours post IV injection mice were sacrificed under isoflurane anesthesia by cardiac perfusion with 1X PBS + Heparin (10 units Heparin per milliliter 1X PBS). Following perfusion, mice were decapitated using a pair of sharp surgical shears and the brain was carefully excised from the cranium. Brains were fixed in 4% PFA for 24hrs then equilibrated in 30% sucrose for 48 hours. Then, mouse brains were sectioned at 50 μm thickness using a freezing sliding microtome. Staining was performed to visualize amyloid plaques using the Congo Red derivative X34 . Tissue P96H-Triplet-647 and H1-Triplet-647 on APP/PS1 mice, brain sections at around 2.5mm posterior to bregma were selected for confocal fluorescence imaging. The brain sections were equally divided into 10 parts in both horizontal (x) and vertical (y) directions . X and y coordinates were randomly selected using by random numbers from 1 to 10. When  coordinates fell onto a cortical area, stacks of images were taken which covered 16μm depth (z direction) starting from the top of the brain section. When  coordinates fell outside of the cortex, the microscope was moved to the next randomized  coordinates without taking images. Brain sections were imaged using a Zeiss LSM 510 microscope. Stacks of images were acquired with eight images per stack and 2μm optical thickness at 20x magnification using a Zeiss Plan-Apochromat 20x/0.8NA lens. Images were acquired using laser wavelength at 633nm to visualize the VHH triplets conjugated with 647 dye and 405 nm wavelength laser to visualize the X-34 dye. For 647 channel images, laser at wavelength 633nm was used with a LP650 filter and laser power set to be at transmission 100%. For X34 channel images, laser at wavelength 405nm was used with a BP420-480 filter and laser power set to be at transmission 15%. The images were exported in lsm file format for image analysis using ImageJ.To objectively evaluate the effect of intravenous injection of M1ex vivo M1P96H-Triplet-647 for fluorescence confocal microscopy were used as a positive control. For ex vivo M1P96H-Triplet-647 staining, naïve brains were sectioned and washed in 1XPBS. Sections were incubated in 3% normal donkey serum for 30min to block non-specific binding. After blocking, tissue sections were incubated overnight at 4C° in M1P96H-Triplet-647 at a concentration of 10ug/ml in blocking solution. Tissues were then washed in 1xPBS three times followed by X34 staining as described.Naïve APP/PS1 positive mouse brain sections stained only with X-34 for fluorescence confocal microscopy were used as a negative control. Naïve APP/PS1 positive mouse brain sections stained with both X34 and To quantitatively analyze the confocal microscope images, thresholding and particle analysis were performed to remove background signals and isolate target structures of interest. The percent area of X34 stained amyloid plaques was quantified with thresholding at  followed by Particle analysis. To remove low signal background, 647 channel image thresholding was set at 0–1000. Then the 647 channel images was analyzed with particle analysis. The parameters for particle analysis were set to be 0–1000 pixels for size and 0.1–1.0 for circularity. The parameters for thresholding and particle analysis were determined through testing of different parameters to capture the qualitative morphology of amyloid plaques as assessed by experienced investigators. Amyloid plaque levels were represented as % area of X34 coverage and VHH triplet-647 entry into the brain parenchyma was represented as % area of Alexa 647 dye coverage in the areas with X34 staining. Because there is mouse-to-mouse variability in plaque size and X34 staining intensity, we analyzed the ratio of the Triplet-647 coverage to X34 coverage.P96H-triplet-647 and H1-triplet-647 to amyloid plaques in APP/PS1 transgenic mice brains. Graphs were created using Prism. Sample sizes were based on availability of transgenic mice and previous experiments. No formal power calculations were performed.Unpaired student t-test were performed to evaluate the differences between the transcytosis ability of M1E. Coli cells [S2 Fig). To avoid potential hypothermia effects caused by endotoxin, endotoxin was removed after purification. After endotoxin removal, the level of endotoxin level in VHHs was less than 0.1 endotoxin unit/ml (EU/ml) within normal range [VHHs contamination by endotoxins is a byproduct of expression in li cells . Endotoxli cells . It was al range .P96H-triplet-NT) had characteristics consistent with expectation.  showed the SDS-PAGE gel and exemplar size-exclusion column results of the VHH monomer-NT and VHH-heterotrimer-NT after purification. VHH monomer-NTs had size around 14kDa  and VHH heterotrimer-NTs had size between 38-49kDa .VHH monomer-NT (M1-NT and H1-NT) and VHH-heterotrimer-NT  and acidic pH (pH = 5.5), lower pH did not alter the dissociation of M1WT to mTfR but substantially impacted the dissociation of M1P96H to mTfR.Histidine mutations could potentially impart pH dependence of binding . HistidiWT-NT, M1P96H-NT, and M1R100dH-NT to mTfR were <1nM, 1.12nM, 1.52nM. While the affinity (KD) of M1AA-NT and H1-NT to mTfR was not detectable. Fig 2 shows the association and dissociation curves of the M1WT-NT, M1P96H-NT, M1AA-NT, M1R100dH-NT and H1-NT to mTfR. M1WT-NT, M1P96H-NT, and M1R100dH-NT bond to mTfR well while M1AA-NT and H1-NT showed no binding. M1WT-NT and M1P96H-NT showed no binding to (Cys89-Phe760) human TfR extracellular domain  .Then the affinity of M1 variants to mTfR was measured using Octet. Based on Octet measurement, the affinity (KD) of M1WT-NT, M1R100dH-NT and M1P96H-NT were injected to WT mice. NT alone was injected into WT mice at the same molarity as an initial negative control. At a dose of 600nmol/kg body weight, the M1 variants with different binding affinities to mTfR show different hypothermia effects . M1WT-NT reduced temperatures by approximately 2C°, with an effect that lasted approximately 2 hours. M1P96H-NT, at the same dose, decreased temperature by 6C° with effects that lasted more than 4 hours, indicating that M1P96H-NT appeared to improve CNS penetration. NT alone did not show any hypothermia effect, indicating that peripherally administered NT alone does not appear to cross the BBB into the CNS. M1R100dH-NT gave less than 2C° of hypothermia effect. Mouse body temperatures were stable at baseline.To assess BBB transcytosis ability, several M1 variants were fused to NT and screened based on the extent of hypothermia effects. Three M1 variant NT fusions, including M1AA-NT that has minimal TfR binding and a different VHH that binds to human TfR called H1 were tested by an investigator blinded to the identity of the injected materials. M1AA-NT and H1-NT were tested at a higher molarity in a previous experiment and showed no obvious hypothermia effects . The blinded experiment results were consistent with the previous finding: M1P96H-NT clearly reduced the temperature the most, with a maximum drop of about 4C°. The other M1-NTs and H1-NT did not reduce the temperature substantially . These results indicate that temperature measurement experiments facilitated the identification of an anti-mTfR VHH variant which induced substantial CNS effects.To confirm and extend these findings, a blinded experiment was performed. The previous three M1 variants, plus a fourth mutant M1P96H-NT injected to mice affects the extent of hypothermia effects, we injected M1P96H-NT to WT mice at four different doses: 67nmol/kg body weight, 200nmol/kg body weight, 600nmol/kg body weight, and 1800nmol/kg body weight. Twelve mice were used in this experiment, with three mice at each dose . At the two lower doses, there was minimal hypothermia effects. With increased M1P96H-NT concentration, the maximum temperature drop increased to about 8C° and also the duration of hypothermia was extended to as long as 5 hours. Thus, these was an apparently monotonic dose-response relationship between intravenous M1P96H-NT and hypothermia.To understand whether the amount of M1P96H was chosen as the variant to use to assess ability to cross the BBB after conjugating to a payload. As a proof of concept, we used the anti-Aβ VHH dimer Nb3-Nb3 as the payload fused in a single polypeptide with M1P96H and NT and named M1P96H-Triplet-NT. The Nb3-Nb3 dimer was also fused to H1 and NT and named H1-Triplet-NT as a negative control. The M1P96H-Triplet-NT and H1-Triplet-NT were injected intravenously into six WT mice (three mice for each Triplet-NT fusion). Body temperature was measured as described in the Temperature measurement section. Fig 6 shows the temperature change after tail vein injection of M1P96H-Triplet-NT and H1-Triplet-NT. For M1P96H-Triplet-NT, there was about 4C° temperature drop after the intravenous injection and the hypothermia effect peaked at about 1 hour post injection. There was no substantial hypothermia effect after intravenous injection of H1-Triplet-NT. This result confirmed that after fusing to an anti-Aβ VHH dimer payload, the M1P96H-Triplet-NT was still able to get across the BBB and exert a CNS effect.Based on the octet results and temperature measurement, M1P96H and Nb3-Nb3 dimer to their targets, the affinity of M1P96H-triplet to mTfR and to Aβ were measured to be 1.63nM and 9.98nM using Octet . The affinity of the M1P96H-triplet to mTfR was on the same order before and after the fusion. The affinity of H1-Triplet-647 to mTfR and Aβ was also assessed using Octet and measured to be no binding to mTfR and 9.44nM to Aβ . It was verified that the fusion of M1P96H and H1 to VHH dimer Nb3-Nb3 did not change their binding affinities.To confirm that fusion with Nb3-Nb3 did not affect the binding ability of M1P96H-triplet binding to mTfR, a mTfR ELISA on M1P96H-triplet before and after Alexa 647 dye labelling was performed. The binding curves before and after labeling overlapped with each other , indicating that the Alexa 647 dye labeling did not affect the binding to mTfR.To ensure that the dye labeling did not affect M1P96H-Triplet-647 and five mice were injected with H1-Triplet-647 as a negative control .VHH-Triplets were injected to APP/PS1 positive transgenic mice to directly assess brain target (Aβ) engagement through confocal microscopy. Ten transgenic mice were randomized into two groups, with five mice in each group. Five mice were injected with M1Figs . Because the NT-induced hypothermia effect was most prominent in the first two hours post injection in previous experiments using NT fusions, transgenic mice were sacrificed two hours post IV injection. Of note, when the VHH-Triplets were fused to NT and labeled with Alexa 647, they did not cause substantial temperature drop , likely because the Alexa 647 labeling blocked the binding of NT to its receptor due to the presence of a lysine residue in the NT peptide. Fig 7 shows exemplar confocal images of brains injected with M1P96H-triplet-647  or H1-triplet-647 . From the exemplar images, it appeared that most of the 647 signals were similar to background, with some areas that had morphology consistent with amyloid plaque labeling . However, surprisingly, the confocal imaging patterns were similar in both M1P96H-triplet-647 and H1-triplet-647 injected mice, suggesting a modest amount of non-specific BBB crossing and Aβ plaque binding plus background autofluorescence, rather than the expected extensive mTfR-mediated transcytosis and Aβ plaque binding.After IV injection, the brain sections were stained with X34 to reveal the location of amyloid plaques . Thus, the modest plaque labeling after intravenous injection with M1P96H-triplet-647 is not likely to be attributed to lack of plaque binding affinity. Autofluorescence was relatively modest in the 647 channel in sections labeled with only X34 .To confirm that the lack of anti-Aβ plaque binding after IV injection by M1P96H-triplet-647 and H1-triplet-647 plaque labeling, we performed randomized, blinded, automated analyses of the brain sections to objectively compare the confocal imaging results. Eleven or twelve confocal image stacks were acquired at randomly selected x, y coordinates in cortex . Confocal images of brain sections were processed using ImageJ for quantitative assessment.Amyloid plaque distribution was not consistent across mice and across regions of brain. To avoid subconscious bias in selecting regions for imaging and to assess for more subtle quantitative differences between M1Materials and Methods, the % area of X34 coverage, % area of Triplet-647 coverage and the % area ratio of Triplet-647/X34 coverage were averaged across the eleven to twelve images stacks acquired for each mouse brain. The average values of % area of each mouse brain was plotted in Fig 9 Comparing mouse brains injected with M1P96H-triplet-647 or H1-triplet-647, the average values of % area of Triplet-647 coverage/% area X34 coverage had no significant difference . There was also no significant difference for % area X34 coverage or % area Triplet-647 coverage .As described in S12 Fig). First, X34 images were alternatively thresholded at  and  and were converted to binary images. Because amyloid-beta pathology can extend beyond the boundaries of X34 positive fibrillar plaque cores, the X34 images thresholded at  were also processed with dilation in ImageJ to add pixels from the edges of plaques. These plaque and peri-plaque regions identified on the X34 images were applied to the 647 channel images to calculate the mean 647 signal of these regions. The mean 647 signals were averaged across image stacks acquired for each mouse brain The average values of mean 647 signals of each brain were plotted in Fig 10 There was no significant difference of the mean 647 signal between the two groups in any of these analyses. These findings confirmed that there was no detectible RMT of M1P96H-triplet-647 into the cortex, despite good evidence for a CNS effect for the same construct when fused to neurotensin.To ensure the finding was not affected by the specific analysis method, confocal images of brain sections were analyzed in several alternative ways using ImageJ (P96H-triplet-647 (S1 File). We found no evidence of plaque labeling using light microscopic immunohistochemistry either .As a further alternative detection method, we used light microscopic immunohistochemistry with an anti-alpaca VHH secondary antibody on brain sections from mice injected with H1-triplet-647 or M1S14 Fig). This indicates that M1 likely binds to a different portion of the mTfR extracellular domain than transferrin and makes it unlikely that transferrin in the blood interferes with the binding of M1 constructs to mTfR at the BBB.In the blood, transferrin is present in high concentrations. To test the hypothesis that the mTfR VHH M1 constructs interacted with transferrin itself during transferrin binding, we tested the affinity of M1 for the extracellular domain of mTfR in the presence or absence of mouse iron bound (holo) transferrin. There was no effect of 10 micrograms per ml of mouse holo transferrin on the affinity of M1 for mTfR  engagement in the future [ex vivo brain sections at sub nM concentration , it might not be sensitive enough to detect the VHHs in vivo. Alternative VHH-triplet detection methods were explored by labeling the VHHs with an anti-alpaca VHH secondary antibody, followed by DAB staining . However, this method did not improve the VHH detection sensitivity. Other staining methods will need to be explored for VHHs detection.Additionally, in this study, the minimum required dose for NT induced temperature effect was about 20-fold higher than Stocki et al. . The rel neurons , it is pe future , 23. AddP96H-NT induced substantial hypothermia, the affinity and kinetics of this anti-mTfR VHH variant have not been fully optimized. Researchers have discovered anti-TfR antibodies with affinities ranging from sub nM to ~100nM which were able to cross the BBB through TfR-mediated transcytosis [Fig 2 shows that the dissociation rate of the M1P96H from mTfR was 1.33X10-4 which could be still too low. Based on Hultqvist et al., after the VHH binds to the TfR and the complex is internalized, the VHH needs to dissociate from the TfR to get released efficiently into the brain parenchyma [P96H-Triplet-NT after dye labeling. This is because Alexa 647 dye was conjugated to M1P96H -Triplet-NT through NHS ester reaction. 647 dye binds to the lysine group on NT and disables NT’s ability to induce hypothermia effect . It is possible that dye modification with Alexa 647 affected BBB transcytosis even though it did not affect mTfR binding affinity in vitro . Alternative labeling strategies will need to be tested in future experiments. Reduction in autofluorescence due to lipofuscin [S14 Fig). Also, it is formally possible, though unlikely, that older APP/PS1 transgenic mice have a less permeable BBB than young WT mice; we have not directly tested hypothermia effects in older APP/PS1 mice. A minor limitation is that the temperature measurement was conducted using an infrared thermometer. Although belly fur was removed to decrease variation and increase the accuracy and precision of temperature measurement, the use of infrared thermometer to measure belly temperature still introduces inter- and intra- user error. Also, the mice need to be anesthetized for acquisition of a steady temperature using the thermometer. Although we kept the anesthesia time short (about 20 sec) each time to minimize the influence of anesthesia on mouse body temperature. The use of anesthesia still introduced extra variations. Other temperature measurement methods such as the use of implanted thermometers which provide real-time temperature readings and eliminate interference with mice should be considered for future experiments. Finally, we acknowledge that only one brain section posterior to bregma was analyzed for each mouse. More brain sections for each mouse which cover additional brain areas, including hypothalamus, will need to be imaged and analyzed in the future.There are several limitations of this study. First, although the M1scytosis , 79–81. scytosis . Karioliscytosis . This 8Dscytosis . Based oscytosis , antibodrenchyma . When thpofuscin  may alsopofuscin . It is pThere are many important future directions for this line of investigation. This manuscript showed that pH-dependent M1 variant P96H improves TfR-mediated BBB transcytosis. Utilizing this property, we are in the process of screening other pH-dependent VHH variants to improve anti-mTfR and anti hTfR-mediated BBB transcytosis.in vitro BBB models have been used. Cecchelli et al. developed a human in vitro BBB model using cord blood-derived hematopoietic stem cells [in vitro predicted ratio of unbound drug concentration in brain and in vivo ratio reported in humans. Shayan et al. made a murine in vitro BBB model using murine brain microvascular endothelial cells which also shows good correlation of compound permeability compared with in vivo values [in vitro models do not always reflect the in vivo BBB function. Garberg et al. evaluated multiple different in vitro models in comparison with an in vivo mouse brain uptake assay to understand the in vitro models’ potential to predict in vivo transport of compounds across BBB [in vitro and in vivo data were obtained with a total of twenty-two compounds. Because of the complexity of the in vivo environment, in vitro BBB models were not recommended to be used in isolation to assess target engagement in brain [Because the NT induced hypothermia effect does not give enough information about target engagement in brain areas like cortex, additional approaches to assess BBB transcytosis are needed. To allow high throughput testing and prediction of therapeutic and diagnostic agent delivery to the CNS, em cells . This moo values . Howeverross BBB . Low corin brain . There ain brain . Yu et ain brain . Other sUsing RMT to transport payload across the BBB has limitations, such as downregulating the targeted receptors and competition with natural ligands which may affect the receptors’ normal physiological functions , 88, 89.E. Coli. and contaminated by endotoxins in the outer membrane of this gram-negative bacteria [In this study, the VHHs were produced in bacteria . Extra sbacteria . Also, tThe reduced size of VHHs does have some disadvantages compared to traditional monoclonal antibodies, such as shortening their blood half-life and reducing total brain exposure , 28. ExtP96H could be conjugated to nanobodies against another widely distributed endogenous target such as the ATP-gated ion channel P2X7, which is widely distributed throughout the brain [P96H-anti P2X7 VHH fusion would then be intravenously injected to mice and its target engagement in cortex and hypothalamus would be assessed and compared. Furthermore, different sizes of dextrans could be intravenously injected to the mice to understand the BBB penetration capacity in hypothalamus and other brain regions [While not directly related to the main aims of the project involving development of brain MRI molecular contrast agents, the mechanisms underlying potential differences in BBB function in the hypothalamus vs. cortex are worthy of further investigation. To begin, target engagement of the Triplet-647 in hypothalamus will need to be assessed. The hypothalamus area of the brain sections will be imaged with confocal microscope and analyzed using ImageJ. Then, the M1 regions .in vivo pharmacokinetics of the contrast agent and optimized the MRI sequence for MR T1 imaging [in vivo imaging. The ability of molecular contrast agents to cross the BBB and bind to brain targets in sufficient quantity to give conspicuous MRI signals will need be tested with in vivo MR imaging in the future.The ultimate goal of this study is to develop a family of molecular MRI contrast agents for diagnosis and assessment of neurodegenerative diseases. Misdiagnosis of neurodegenerative diseases is common because of their heterogeneous nature . Early d imaging , 99, but imaging . Silleru imaging . HoweverP96H, was identified with good performance in inducing hypothermia, an effect which requires crossing the BBB. This M1 variant was fused to the anti-Aβ VHH dimer and labeled with Alexa 647. Surprisingly, however, the dye labelled VHH did not show detectible labeling of amyloid plaques compared with controls after intravenous injection into transgenic mice. Reasons for the lack of correspondence between NT-induced hypothermia effect and Aβ visualization will need to be explored. Additional methods to assess VHH BBB transcytosis will need to be developed for screening VHHs to facilitate the development of MRI molecular contrast agents.This study used a NT based modular system to screen anti-mTfR VHHs for TfR-mediated transcytosis across BBB. A M1 variant, M1S1 Figbiorender.com.This schematic graph shows the logic of picking coordinates for confocal images acquisition. The right hemisphere of the brain was equally divided into ten parts in both x and y directions.  coordinates were randomly generated using a random number generator. The microscope stage was moved to the target  coordinates using the stage rulings. When  coordinates fell into the areas of cortex, images were taken and cropped to include only cortical areas. When  coordinates fell out of the cortex the coordinates were discarded and no images were taken. Source of the mouse brain: (TIF)Click here for additional data file.S2 FigWT-NT preparations from E. coli decreased hypothermia effects at time 30min, 1hr and 1.5hr after IV injection of the M1WT -NT (n = 3 per group). The maximum hypothermic effect of 600nmol/kg body weight of M1WT -NT was approximately 2C° less after endotoxin removal.Endotoxin removal from in M1(TIF)Click here for additional data file.S3 FigThe purity of the monomer VHH by (a) SDS-PAGE gel and exemplar (b) size-exclusion chromatography over a Superdex75 column for M1WT-NT indicating >95% purity and homogeneity of the material.(TIF)Click here for additional data file.S4 FigP96H-Triplet-NT indicating >95% purity and homogeneity of the material.The purity of the triplet VHH by (a) SDS-PAGE gel and exemplar (b) size-exclusion chromatography over a Superdex75 column for M1(TIF)Click here for additional data file.S5 FigWT, (b) M1P96H and (c) M1R100dH were incubated on mTfR coated ELISA plates, followed by a stringent wash with 1x PBS buffer at pH 7.2 or pH 5.5. Following pH dependent washing, the bound VHH was detected with an anti-alpaca-peroxidase antibody and the reaction terminated by addition of 1M HCl. Error bars represent the standard deviation of the mean values at each data point.M1 variants (a) M1(TIF)Click here for additional data file.S6 FigmTfR and hTfR ELISA on M1WT-NT (a) and M1P96H-NT (b). VHHs were incubated with mTfR or hTfR absorbed to ELISA plates and detected using an anti-alpaca-peroxidase antibody to determine the impact of fluorophore conjugation. The curves show no measurable binding of M1WT-NT (a) and M1P96H- NT (b) to human TfR extracellular domain.(TIF)Click here for additional data file.S7 FigAA-NT and H1-NT were injected at doses of 1400nmol/kg body weight. M1AA-NT and H1-NT lack prominent hypothermia effects (n = 3 per group).M1(TIF)Click here for additional data file.S8 FigP96H-triplet-NT association and dissociation curve to (a) mTfR and (b) Aβ. The affinity of M1P96H-triplet-NTwas measured to be 1.63nM to mTfR and 9.98nM to Aβ. H1-triplet-NT association and dissociation curve to (c) mTfR and (d) Aβ. The affinity of H1-triplet-NT was measured to be 9.44nM to Aβ and no binding to mTfR. (n.b. = no binding).Using biolayer interferometry on an Octet Red96 system, association and dissociation rates were determined by immobilizing biotinylated VHH  or biotinylated amyloid beta  onto streptavidin-coated optical sensors. M1(TIF)Click here for additional data file.S9 FigVHH was incubated with mTfR absorbed to ELISA plates and detected using an anti-alpaca-peroxidase antibody to determine the impact of fluorophore conjugation. The near overlapping binding curves indicate a lack of effect following fluorophore labelling. Error bars represent the standard deviation of the mean values at each data point.(TIF)Click here for additional data file.S10 FigP96H-Triplet-NT-647 was injected at dose of 600nmol/kg body weight (n = 3). M1P96H-Triplet-NT-647 did not cause temperature drop.M1(TIF)Click here for additional data file.S11 FigP96H-Triplet-647 which labels the amyloid plaques. The representative 647 channel microscopy images of the ex vivo brain sections show substantial amyloid plaques labeling with M1P96H-Triplet-647 at concentration of 22.2nM, 2.22nM, and 0.22nM.Mice brain sections were stained with M1(TIF)Click here for additional data file.S12 FigP96H-Triplet-647 labeling was used as the example. Column a. shows a representative confocal microscope image of cortex X34 staining. Column b. shows the X34 labeled images after thresholding. Column c. shows automated selection of regions of plaques based on the thresholded images. Column d. shows the application of selected areas (from thresholded X34 images) to the 647 channel images.An APP/PS1 positive naïve brain slice with ex vivo M1(TIF)Click here for additional data file.S13 FigP96H-triplet-647 (b). Scale bar is 200 μm.Anti-alpaca VHH secondary antibody against triplet-647 was used for immunohistochemistry. Mouse brains injected with H1-triplet-647 (a) and M1(TIF)Click here for additional data file.S14 FigNo change in binding affinity of mTfRnb-M1 to mTfR in the presence or absence of 10ug/ml of. Anti-mTfR VHH M1 was incubated with mTfR absorbed to ELISA plates and detected using an anti-alpaca-peroxidase antibody to determine the impact of mouse transferrin presence. The binding curves nearly overlap, indicating the presence or absence of mouse transferrin did not affect the binding affinity of M1 to mTfR. Error bars represent the standard deviation of the mean values at each data point.(TIF)Click here for additional data file.S1 FileStaining was performed to visualize amyloid plaques using the Anti-alpaca VHH secondary nanobody. Free-floating 50 μm tissue was rinsed with PBS for three times. Then, tissues were incubated in 2% normal goat serum (NGS) with 0.1% Triton X-100 in PBS for 60min to block non-specific binding. After blocking, tissue was rinsed with 1X PBS for three times then incubated in 0.5% NGS with 0.1% Triton X-100 in PBS with 0.8mg/ml Anti-alpaca VHH secondary antibody  for 60min. After antibody incubation, the tissue was rinsed with PBS for three times, followed by incubation with ABC Elite (Vector Laboratories) at a 1:400 dilution in PBS for 60min. The tissue was then rinsed with PBS for three times and developed with 3,3’-diaminobenzidine . The tissue was rinsed with 1X PBS for three times then mounted onto positively charged slides . The mounted tissue sections were allowed to dry overnight at room temperature and dehydrated using ethanol series. Then, the sections were cover slipped using VectaMount® Permanent Mounting Medium (H-5000-60).(PDF)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file.


ARID1A and MYD88 mutations, and RET subtype had increased KMT2D, LYN and SOCS1 mutations. RES and RET subtypes showed significant enrichment in oncogenic pathways, such as ERK, NF-κB, JAK-STAT, and immune-associated pathways. As for tumor microenvironment, RES subtype exhibited increased recruiting activity of CD8 + T, T helper 1, and natural killer cells, and RET subtype with increased recruiting activity of CD4 + T and regulatory T cells in silico. There was a positive correlation between transcripts of IL-2R and immune checkpoint expressions including PD-1 and CTLA-4. Our findings identified that dynamic change of sIL-2R, with this simple and easy detection method in peripheral blood, had long-term prognostic effect and specific relation to microenvironment alterations in DLBCL.Diffuse large B-cell lymphoma (DLBCL) is an aggressive lymphoma with variable clinical outcomes and prediction of prognosis remains important for long-term remission. We performed serial serum soluble interleukin-2 receptor (sIL-2R) measurement pretreatment and before each cycle of the treatment in 599 patients with de novo DLBCL. Genomic and transcriptomic features were analyzed by 223 DNA- and 227 RNA-sequencing, respectively. Applying the cut-off value to sIL-2R pretreatment and cycle 2 (C2) level, patients were classified into FINE subtype (pretreatment low level) with good prognosis, RES subtype (pretreatment high level and C2 low level) with intermediate prognosis, and RET subtype (pretreatment high level and C2 high level) with poor prognosis, independent of International Prognostic Index. In “others” genetic subtype, dynamic change of sIL-2R showed prognostic significance and genetic features. Compared with FINE subtype, RES subtype had increased The online version contains supplementary material available at 10.1186/s40364-022-00401-4. To the Editor,N = 379), RES subtype with pretreatment high and C2 low level (N = 148), and RET subtype with pretreatment high and C2 high level (N = 72). The serial sIL-2R measurement was displayed in Fig. SDiffuse large B-cell lymphoma (DLBCL) is the most common subtype of lymphoma with clinical and molecular heterogeneity . Due to evel N = 48, and Revel N = 48, and Revel N = 48, and RIn both training cohort Fig. a and valARID1A and MYD88, and RET subtype had a higher mutation frequency in KMT2D, LYN, and SOCS1  subtypes of DLBCL demonstrated prognostic significance and therapeutic implications . Whole eRNA sequencing was screened in 227 patients, including 126 FINE, 69 RES and 31 RET subtypes. With similar pattern of pathway alterations in the RES and RET subtypes Table S, we compTo our knowledge, this is the first study to evaluate dynamic change of sIL-2R on prognostic significance and tumor microenvironment in DLBCL. With better understanding of sIL-2R biology profile, more clinical trials targeting sIL-2R vulnerability with surveillance of sIL-2R are warranted.Additional file 1: Supplementary Methods. Figure S1. Dynamic change of sIL-2R in DLBCL. Figure S2. Univariate and multivariate risk models in DLBCL according to sIL-2R dynamic change. Figure S3. Survival analysis in DLBCL according to sIL-2R dynamic change in patients risked by R-IPI. Figure S4. Genetic and lymphoma microenvironment features of sIL-2R subtypes. Table S1. Clinical and pathological characteristics of DLBCL patients. Table S2. Multivariate analysis and C-index for progression-free survival (PFS) and overall survival (OS) in DLBCL. Table S3. Pathway alterations in RES subtype. Table S4. Pathway alterations in RET subtype.