Hypercalcemia is a frequently encountered electrolyte abnormality with a well-described differential diagnosis and classic algorithm for evaluation. The treatment for hypercalcemia is dependent on the underlying etiology. Hypervitaminosis D is an uncommon cause of hypercalcemia, but the use of vitamin D supplementation has expanded and case reports of supplemental vitamin D induced hypercalcemia have become more frequent. We present a case of hypervitaminosis D-induced altered mental status where diagnosis was delayed and additional invasive testing was performed due to an assumption regarding phosphatemia.  Vitamin D is a steroid hormone crucial for both bone health and calcium homeostasis . It can 2 95%. Physical exam revealed bilateral upper extremity tremors and obtundation. Pupils were symmetric and reactive to light, and he periodically tracked with his eyes. Neurological exam, including reflexes, was limited due to altered mental status. Cardiopulmonary exam was benign. Chest X-ray was notable for a normal cardiopulmonary silhouette. Electrocardiogram revealed normal sinus rhythm with a QTc interval of 409 ms. Computed tomography of the brain without contrast showed normal gray-white matter differentiation. A 64-year-old man with a history of chronic obstructive pulmonary disease and ethanol abuse use presented to the emergency department with altered mentation. He was obtunded on arrival, unable to provide any additional history. Presenting vitals were notable for blood pressure of 150/93, pulse of 69 beats per minute, temperature of 97.3 °F (36.3 °C), respiratory rate of 18 BPM, and SpOLaboratory evaluation revealed a sodium 143 mEq/L (135 – 145 mEq/L), potassium 3.0 mEq/L (3.6 – 5.1 mEq/L), chloride 105 mEq/L (98 – 110 mEq/L), bicarbonate 27 mEq/L (22 – 32 mEq/L), blood urea nitrogen 14 mg/dL (6 – 24 mg/dL), creatinine 1.11 mg/dL (0.64 – 1.27 mg/dL), undetectable blood ethanol level, blood glucose 95 mg/dL (67 – 99 mg/dL), calcium 12.8 mg/dL (8.5 – 10.5 mg/dL), ionized calcium 6.3 mg/dL (4.2 – 5.2 mg/dL), phosphorus of 2.2 mg/dL (2.4 – 4.8 mg/dL), and albumin 4.0 g/dL (3.5 – 5.0 g/dL). Initial management consisted of volume expansion with 3 L normal saline without improvement in mentation. Calcitonin was given on hospital day 2 and zoledronic acid was given on hospital day 3, but hypercalcemia persisted. Further labwork returned with intact parathyroid hormone (iPTH) 17 pg/mL (18 – 80 pg/mL), parathyroid hormone related peptide (PTHrP) 9 pg/mL (pg/mL 14 – 27), serum protein electrophoresis (SPEP) and immunofixation (IFE) without evidence of monoclonal protein, κ light chains 85.4 mg/L (3.3 – 19.4 mg/L), λ light chains 40.8 mg/L (5.7 – 26.3 mg/L), and β-2 microglobulin 3.75 mg/L (0.97 – 2.64 mg/L). The elevation of κ/λ ratio in the setting of a normal glomerular filtration rate prompted evaluation for a monoclonal gammopathy. Skeletal survey was negative for lytic lesions and bone marrow biopsy was negative for plasma cell dyscrasia. Finally, the following labs resulted: 25-hydroxy vitamin D > 150 ng/mL (30.0 – 100.0 ng/mL), 1,25-hydroxy vitamin D 36 pg/mL (18 – 72 pg/mL). The patient remained agitated and confused for the first 10 days of hospitalization, but mentation improved thereafter with calcium normalizing after 18 days. Upon recovery, he reported misunderstanding medication instructions and had been consuming 4 vitamin D 50,000 IU tablets daily. This case highlights the risks of pathophysiologic assumptions. The findings of hypercalcemia with appropriately suppressed iPTH and low PTHrP resulted in an evaluation for hypervitaminosis D. However, hypophosphatemia directed the evaluation away from hypervitaminosis D, leading to additional evaluation for paraproteinemia. The finding of an abnormal κ-to-λ ratio and elevated β-2 microglobulin, resulted in evaluation for plasma cell dyscrasia with a bone marrow biopsy. The patient had an elevated vitamin D 25OHD and normal calcitriol revealing the ultimate diagnosis to be hypervitaminosis D. The presentation of hypercalcemia frequently varies with the level of serum calcium. Individuals with mild hypercalcemia (< 12 mg/dL) are typically asymptomatic . ModeratHypervitaminosis D classically presents as hypercalcemia with hyperphosphatemia and low PTH , 12. VitSerum phosphorus regulation is likely incompletely understood. Phosphate is regulated by PTH, fibroblast growth factor 23 (FGF23), and 1,25OHD . PhosphaThis case highlights the importance of having a broad differential for hypercalcemia. We recommend that hypervitaminosis D be considered in patients with hypercalcemia and low PTH regardless of phosphorus level. The authors did not receive support for this work. The authors declare no conflict of interest.


Litylenchus crenatae mccannii (Anguinidae) nematodes by systematically testing thermostable polymerases, proofreading enzymes and buffers. The combination of thermostable DreamTaq™, proofreading Pfu polymerase, and PicoMaxx™ buffer provided the best results. These nematodes are the subject of surveys currently active at many sites in the northeastern United States. This new, optimized PCR protocol will be useful for diagnostic labs associated with the surveys.Generating DNA markers for microscopic plant parasitic nematodes can be especially difficult if only a few valuable, tiny specimens are available. Providing a reliable maximum amount of unambiguous genetic information from single nematodes is especially important when identifying damaging, regulated nematodes of importance to trade where a few nucleotide differences in diagnostic markers are significant. There are many possible reasons for difficulty amplifying unpurified nematode DNA for long range PCR followed by direct sequencing. Specimen age, proofreading errors and reagent compatibility during PCR are among those problems. While unsuccessful direct amplification of difficult samples may sometimes be overcome by cloning, a more expensive and time-consuming process. Therefore, long segment PCR of a large 3.5 kb segment of ribosomal DNA was optimized for individual difficult-to-amplify young  Fagus grandifolia) trees in Ohio, Pennsylvania, New York and Connecticut. It was discovered first in 2012 near Lake Erie in the Cleveland Metroparks, region of Ohio (Beech leaf disease (BLD) is an emerging tree disease in the Northeast for American beech . Their ribosomal DNA (rDNA) loci were amplified by PCR with the one primer set and an enhanced DNA polymerase system, and the resulting 3.5 kb  rDNA amplicons were directly sequenced  and PicoMaxx™ High Fidelity PCR System  were selected and tested in this study.Commonly used o 2.9 kb . It has R system . As a reLitylenchus specimens were isolated from the banding lesions of American beech leaves with BLD as described in Litylenchus and the visualization, cleanup and direct DNA sequencing, including sequencing primers, of the PCR products were performed by using the procedures described in previous studies  were performed by using the procedures, including primer sets, 18S-CL-F3 and 28S-CL-R for the 3.5 kb ribosomal loci, 18S-CL-F3 and 18S-CL-R7 for the 18 S locus (1.7 kb) and ITS-CL-F2 and 28S-CL-R for the ITS-28S (D1D2D3) loci (1.9 kb) described in the previous study   was carried out in a 25 μl of mixture containing Platinum™ Taq (10 units/μl) 0.125 μl, 10X PCR Buffer Mg 2.5 μl, MgCl2 (50 mM) 1 μl, dNTP (2.0 mM each) 2.5 μl, Template DNA 2 μl, forward primer (10 μm) 0.75 μl and reverse primer (10 μm) 0.75 μl for either primer set 18S-CL-F3 and 28S-CL-R or ITS-CL-F2 and 28S-CL-R, and molecular biology grade water  15.375 μl. The thermal cycling program was one cycle of 95°C for 3 min; 36 cycles of 95°C for 30 sec, 50°C for 45 sec, 72°C for 3 min; and final extension at 72°C for 7 min.Each PCR amplification with Platinum™ Taq2000™ DNA Polymerase  was carried out in a 25 μl mixture containing Taq2000™ (5 units/μl) 0.25 μl, 10X PCR Buffer 2.5 μl, MgCl2 (50 mM) 0.25 μl, dNTP (2.0 mM each) 2.5 μl, Template DNA 2 μl, both forward primer (10 μm) 0.75 μl and reverse primer (10 μm) 0.75 μl for either primer set 18S-CL-F3 and 28S-CL-R or ITS-CL-F2 and 28S-CL-R, and molecular biology grade water  16 μl. The thermal cycling program was one cycle of 95°C for 3 min; 36 cycles of 95°C for 30 sec, 50°C for 45 sec, 72°C for 3 min; and final extension at 72°C for 7 min.Each PCR amplification with Taq™ DNA Polymerase  alone or combined with DreamTaq™ was carried out in a 25 μl of mixture containing TaKaRa Ex Taq™ (5 units/μl) 0.125 μl (or plus DreamTaq™ (5 units/μl) 0.125 μl), 10X Ex Taq Buffer 2.5 μl, dNTP (2.5 mM each) 2 μl, Template DNA 2 μl, forward primer 18S-CL-F3 (10 μm) 1.25 μl, reverse primer 28S-CL-R (10 μm) 1.25 μl, and molecular biology grade water  15.875 μl (or 15.75 μl). The thermal cycling program was: one cycle of 98°C for 30 sec; 36 cycles of 98°C for 10 sec, 68°C for 5 min; and final extension at 72°C for 7 min.Each PCR amplification with TaKaRa Ex Taq™ was carried out in a 25 μl of mixture containing PicoMaxx™ high fidelity PCR system (PicoMaxx™ (5 units/μl)) 0.5 μl (or plus DreamTaq™ (5 units/μl) 0.125 μl), 10× PicoMaxx™ reaction buffer (PicoMaxx™ buffer) 2.5 μl, dNTP (25 mM each) 0.2 μl, Template DNA 2 μl, forward primer 18S-CL-F3 (10 μm) 1.25 μl, reverse primer 28S-CL-R (10 μm) 1.25 μl, and molecular biology grade water  17.3 μl (or 17.175 μl). The thermal cycling program was one cycle of 95°C for 2 min; 36 cycles of 95°C for 30 sec, 55°C for 45 sec, 72°C for 5 min; and final extension at 72°C for 7 min.Each PCR amplification with PicoMaxx™ High Fidelity PCR System (PicoMaxx™ System)  alone or combined with Dreampfu DNA polymerase  alone or combined with DreamTaq™ was carried out in a 25 μl of mixture containing pfu (2.5 units/μl) 0.75 μl (or plus DreamTaq™ (5 units/μl) 0.125 μl), 10× Pfu reaction buffer, 10× PicoMaxx™ buffer, or 10× DreamTaq™ buffer 2.5 μl, dNTP (25 mM each) 0.2 μl, Template DNA 2 μl, forward primer 18S-CL-F3 (10 μm) 1.25 μl, reverse primer 28S-CL-R (10 μm) 1.25 μl, and molecular biology grade water  16.85 μl (or 16.725 μl). The thermal cycling program was one cycle of 95°C for 2 min; 36 cycles of 95°C for 30 sec, 55°C for 45 sec, 72°C for 5 min; and final extension at 72°C for 7 min.Each PCR amplification with Pwo DNA polymerase  alone or combined with DreamTaq™ was carried out in a 25 μl final volume consisting of two mixtures: 12.5 μl of mixture A containing Pwo (5 units/μl) 0.125 μl (or plus DreamTaq (5 units/μl) 0.125 μl), 10× Pwo reaction buffer or 10× PicoMaxx™ buffer 2.5 μl, and molecular biology grade water  9.875 μl (or 9.75 μl); 12.5 μl of mixture B containing dNTP (25 mM each) 0.4 μl, template DNA 2 μl, forward primer 18S-CL-F3 (10 μm) 1.25 μl, reverse primer 28S-CL-R (10 μm) 1.25 μl. The thermal cycling program was one cycle of 95°C for 2 min; 36 cycles of 95°C for 30 sec, 57°C for 45 sec, 72°C for 5 min; and final extension at 72°C for 7 min.Each PCR amplification with Each PCR amplification with Herculase® II Fusion DNA polymerase  was carried out in a 25 μl of mixture containing Herculase® II Fusion DNA polymerase 0.5 μl, 5× reaction buffer 5 μl, dNTP (25 mM each) 0.25 μl, Template DNA 2 μl, forward primer 18S-CL-F3 (10 μm) 0.625 μl, reverse primer 28S-CL-R (10 μm) 0.625 μl, and molecular biology grade water  16 μl. The thermal cycling program was: one cycle of 95°C for 2 min; 36 cycles of 95°C for 20 sec, 55°C for 20 sec, 72°C for 2 min 15 sec; and final extension at 72°C for 7 min.Each PCR amplification with Phusion™ High-Fidelity DNA Polymerase  was carried out in a 25 μl of mixture containing Phusion™ High-Fidelity DNA Polymerase (2 units/μl) 0.25 μl, 5× reaction buffer 5 μl, dNTP (2.5 mM each) 0.5 μl, Template DNA 2 μl, forward primer 18S-CL-F3 (10 μm) 1.25 μl, reverse primer 28S-CL-R (10 μm) 1.25 μl, DMSO 0.25 μl, and molecular biology grade water  14.5 μl. The thermal cycling program was: one cycle of 95°C for 2 min; 36 cycles of 95°C for 20 sec, 55°C for 20 sec, 72°C for 2 min 15 sec; and final extension at 72°C for 7 min.Taq™ system were carried out in 10 out of 11 Fall specimens. The direct sequencing for the three loci (3.5 kb) was also conducted successfully in all specimens, except for 104H89 and 104H90 with low PCR yields that were good for sequencing only one or two loci. The 3.5 kb rDNA sequences generated for the specimens, 104H82 ( MN525396) and 104H83 (MN525397) were submitted to GenBank. This result shows that DreamTaq™ had the ability to amplify the 3.5 kb target in most Fall specimens within the size limit by Taq DNA polymerase up to 3 to 4 kb on amplicon , and ITS and 28 S loci (1.9 kb) within the 3.5 kb target, respectively. The amplifications showed that DreamTaq™ can amplify both medium 1.7 kb and 1.9 kb fragments with high yield  seen in Both TaKaRa  compare  with 3B.pecimens , the Drebination . This suPwo (derived from Pyrococcus woesei), another proofreading DNA polymerase, was tested in line with the Pfu in PicoMaxx™ buffer. Pwo or DreamTaq™ alone . No significant amplifications of the 3.5 kb target were seen in the presence of either buffer (data not shown). This confirms again that PCR buffer is another key to the success of the DreamTaq™ and Pfu or Pwo combination.with Pwo . The preTaq® system and PicoMaxx™ system was also performed. Taq® systems failed to amplify the 3.5 kb target, but the PicoMaxx™ system gave DreamTaq™ dramatic leverage over the TaKaRa Ex Taq® system  and ability to amplify long target (>20 kb) (both Agilent and Thermo Fisher Scientific web sites). Herculase® II Fusion DNA polymerase and Phusion™ High-Fidelity DNA Polymerase were tested. Fusion DNA polymerase is an engineered fusion of a proofreading polymerase and a processivity-enhancing domain  and offeTaq2000™, which is one of the components of the PicoMaxx™ system, Platinum™ Taq and DreamTaq™ were also compared in Spring specimens. In the presence of their own buffers, both the long segment PCR for the 3.5 kb target and the medium range PCR for the 1.9 kb target were carried out. In the long segment PCR amplifications, all of the three Taqs failed to amplify the 3.5 kb target  and DreamTaq™ can successfully amplify the 3.5 kb target in the specimens where both DreamTaq™ and PicoMaxx™ systems failed separately.The PCR performances of b target . In the Taq-based blend systems, TaKaRa Ex Taq® DNA Polymerase in the specimen 104J58 (OH), PicoMaxx™ High Fidelity PCR System in the Summer Specimen 104K17 (OH); by the combination of DreamTaq™ and PicoMaxx™ High Fidelity PCR System in the Summer specimens, 104K25  and 104K37  were sequenced and the resulting rDNA sequences (ITS and 28 S loci) were deposited in GenBank with the accession numbers, 104H82, MN525396; 104H83, MN525397; 104J58 MN525398; 104K17, MN525399; 104k25, MN525400; 104K37, MN525401, respectively. Multiple alignments of these sequences above with the 3.5 kb rDNA (MK292137 and MK292138) of the Ohio Litylenchus specimens in the previous study  in these Summer specimens . DreamTaq™ also demonstrated its higher sensitivity than either Taq2000™ or Platinum™ Taq , Pristionchus sp. (Rhabditida) and Prodorylaimus sp. (Dorylaimida) when their specimens were difficult to amplify with the one primer set and DreamTaq™ (data not shown). This improvement provides high fidelity, sensitivity and yield with minimum optimization of reaction and cycling conditions. It should not be limited to long segment PCR amplification only, and could be considered for short range PCR with forensic or ancient DNA, single copy nuclear gene PCR or where improved proofreading can rescue mismatches that take place between the 3’ primer termini and its target templates.Taken together, the size limit to the 3.5 kb target by


The title compound exhibits exceptionally weak intermolecular C—H⋯π hydrogen bonding of the ethynyl groups, with the corresponding H⋯π separations [2.91 (2) and 3.12 (2) Å] exceeding normal vdW distances. This bonding compliments distal contacts of the CH ⋯π type [H⋯π = 3.12 (2)–3.14 (2) Å] to sustain supramolecular layers. 14H16, exhibits exceptionally weak intermolecular C—H⋯π hydrogen bonding of the ethynyl groups, with the corresponding H⋯π separations [2.91 (2) and 3.12 (2) Å] exceeding normal vdW distances. This bonding complements distal contacts of the CH ⋯π type [H⋯π = 3.12 (2)–3.14 (2) Å] to sustain supramolecular layers. Hirshfeld surface analysis of the title compound suggests a relatively limited significance of the C⋯H/H⋯C contacts to the crystal packing (24.6%) and a major contribution from H⋯H contacts accounting 74.9% to the entire surface.The title compound, C The shortest contacts between successive layers concern interactions involving the methylene groups C10H⋯Cg(C1C2)iv  . The Hirshfeld surface of the molecule, mapped over dnorm in the color range 0.0957 to 1.3378 a.u., indicates only a set of normal vdW contacts (white regions) corresponding to the closest interactions  are by far the major contributors (74.9%) to the entire surface, while the fraction of C⋯H/H⋯C contacts accounts for only 24.6%. The latter value may be compared with contributions of 40.0 and 32.4% calculated for α,ω-octa- and decadiynes  global, I. DOI: 10.1107/S2056989020005964/lh5958Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989020005964/lh5958Isup3.cmlSupporting information file. DOI: 2000259CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the molecular packing, O—H⋯O and O—H⋯S hydrogen bonds lead to supramolecular layers propagating in the ab plane.In the title tri-substituted thiourea molecule, a substantial twist is evident as seen in the dihedral angle of 65.92 (12)° between the planes through the CN 12H15N3O5S, a trisubstituted thiourea derivative, the central CN2S chromophore is almost planar (r.m.s. deviation = 0.018 Å) and the pendant hydroxyethyl groups lie to either side of this plane. While to a first approximation the thione-S and carbonyl-O atoms lie to the same side of the molecule, the S—C—N—C torsion angle of −47.8 (2)° indicates a considerable twist. As one of the hydroxyethyl groups is orientated towards the thioamide residue, an intramolecular N—H⋯O hydrogen bond is formed which leads to an S(7) loop. A further twist in the molecule is indicated by the dihedral angle of 65.87 (7)° between the planes through the CN2S chromophore and the 4-nitrobenzene ring. There is a close match between the experimental and gas-phase, geometry-optimized (DFT) molecular structures. In the crystal, O—H⋯O and O—H⋯S hydrogen bonds give rise to supramolecular layers propagating in the ab plane. The connections between layers to consolidate the three-dimensional architecture are of the type C—H⋯O, C—H⋯S and nitro-O⋯π. The nature of the supramolecular association has been further analysed by a study of the calculated Hirshfeld surfaces, non-covalent interaction plots and computational chemistry, all of which point to the significant influence and energy of stabilization provided by the conventional hydrogen bonds.In the title compound, C Such N,N′-di-N′-benzoylthiourea derivatives have a carbonyl group connected to the thiourea framework and offer opportunities for rich coordination chemistry as these molecules feature both hard (oxygen) and soft (sulfur) donor atoms along with nitrogen donors and indeed, a variety of coordination modes have been observed. The neutral molecule has been observed to coordinate in a monodentate-S mode  interactions ° is consistent with a significant twist in the molecule about the C1—N2 bond; the O3—C6—N2—C1 torsion angle is −3.6 (2)°.Selected geometrical data for (I)2S plane (r.m.s. deviation = 0.017 Å). Crucially, the O1-hydroxyethyl group is folded towards the thioamide residue, which allows for the formation of an intramolecular N2—H⋯O1 hydrogen bond and an S(7) loop, Table 22S atoms and the terminal C7–C12 aryl ring. From Table 1The hydroxyethyl groups lie to either side of the CNd,p) basis set ° (X-ray) versus −16.5° , indicating a greater deviation from the anti-disposition in the optimized structure. Also, the N1—C2—C3—O1 and N1—C4—C5—O2 torsion angles are close to symmetric in the optimized structure cf. the experimental structure. Similar trends were noted in analogous calculations performed on the 4-methyl analogue . The layers are connected into a three-dimensional architecture by methylene-C—H⋯O(carbonyl), methylene-C—H⋯S(thione) and comparatively rare nitro-O⋯π(aryl) contacts, Fig. 3b).In the crystal of (I)s Table 2 lead to Crystal Explorer 17  reflects the relative likelihood of the formation of X-to-Y interactions in a crystal, i.e. the ratio between the proportion of actual contacts in a crystal to the theoretical proportion of random contacts. Data for (I)The enrichment ratio (ER) descriptor, which is derived from the analysis of the Hirshfeld surface (Jelsch a)–(f), respectively, with a summary of the percentage contributions from the various contacts given in Table 5de + di ∼1.8 Å in Fig. 7c). The prominent features in Fig. 7d) reflect the significant C⋯H/H⋯C contacts evident in the packing, Tables 2de + di ∼2.1 Å in the fingerprint plot of Fig. 7e). The 5.8% contribution from C⋯O/O⋯C contacts and the aforementioned ER value of 1.66 clearly indicate the significance of the nitro-N—O⋯π interaction upon the packing; this interaction is reflected in the pair of short spikes de + di ∼3.0 Å, Fig. 7f).The overall fingerprint plots for (I)EBSSEint) are listed in Table 6et al., 2019The energy calculations were performed using DFT-wB97XD/aug-cc-pVTZ  of all pairwise interactions sums to −45.89 kcal/mol, while the total dispersion energy term (Edispersion) computes to −51.51 kcal/mol.From the aforementioned, the molecular packing is clearly governed by directional hydrogen bonding between molecules. The simulated energy frameworks N(H)C(=S)N(CH2CH2OH)2, namely Y = H, which has been reported twice , 68.96 (12), 69.51 (8) and 72.15 (10)° for (I)Y = H, F and Me, respectively.There are three literature precedents to (I)The molecular packing in the crystals is also very similar with the formation of the intramolecular thioamide-N—H⋯O(hydroxy) hydrogen bond as well as the intermolecular hydroxy-O—H⋯O(hydroxy) hydrogen and hydroxy-O—H⋯S(thione) hydrogen bonding, leading to a supramolecular layer in each case.−1): 3277 , 3170 , 3077 , 2973–2882 , 1692 , 1538 , 1524 , 1343 , 1270 , 1053 , 734 . UV : λmax nm (log ∊) 366.4 (4.16), 301.6 (4.88), 271.2 (5.00), 205.8 (5.14).Synthesis of (I)2, equivalent a 15% weight loss, in the first stage in the range 194 and 222°C. This was followed by the liberation of a benzene molecule, corresponding to 29% weight loss, between 222 and 282°C, whereas the subsequent stages involve the pyrolysis of CO (282 to 360°C) and OH (360 to 496°C) corresponding to 15 and 11% weight loss, respectively. Gradual weight loss continued beyond 800°C.The pyrolytic process  for (I)Uiso(H) set to 1.2Ueq(C). The O- and N-bound H atoms were located from a difference map and refined with O—H and N—H = 0.84±0.01 and 0.88±0.01 Å, respectively, and with Uiso(H) = 1.5Ueq(O) and 1.2Ueq(N).Crystal data, data collection and structure refinement details are summarized in Table 710.1107/S2056989019017328/hb7880sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989019017328/hb7880Isup2.hklStructure factors: contains datablock(s) I. DOI: 1919879CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The title morpholinochlorin adopts a ruffled conformation of its porphyrinic π-system chromophore inducing a red-shift of its optical spectrum compared to its chlorin analog. 46H16F20N4O3, was crystallized from hexane/methylene chloride as its 0.44 methylene chloride solvate, C46H16F20N4O3·0.44CH2Cl2. The morpholinochlorin was synthesized by stepwise oxygen insertion into a porphyrin using a ‘breaking and mending strategy’: NaIO4-induced diol cleavage of the corresponding 2,3-dihydroxychlorin with in situ methanol-induced, acid-catalyzed intramolecular ring closure of the intermediate secochlorins bisaldehyde. Formally, one of the pyrrolic building blocks was thus replaced by a 2,3-dimethoxymorpholine moiety. Like other morpholinochlorins, the macrocycle of the title compound adopts a ruffled conformation, and the modulation of the porphyrinic π-system chromophore induces a red-shift of its optical spectrum compared to its corresponding chlorin analog. Packing in the crystal is governed by interactions involving the fluorine atoms of the pentafluorophenyl substituents, dominated by C—H⋯F interactions, and augmented by short fluorine⋯fluorine contacts, C—F⋯π interactions, and one severely slipped π-stacking interaction between two pentafluorophenyl rings. The solvate methylene chloride molecule is disordered over two independent positions around an inversion center with occupancies of two × 0.241 (5) and two × 0.199 (4), for a total site occupancy of 88%.The title morpholinochlorin, C Those adjacent to the morpholine moiety (C6F5 rings of C21 and C39) are more sterically encumbered and are about 10° closer to perpendicular to the macrocycle plane, with values of 82.70 (3) and 81.44 (2)°. The corresponding values for compound 1b are very similar, with values of 71.89 and 73.73°, and 89.55 and 86.32°, respectively.Similar to other 1b and 1d allows us to investigate how minor conformational changes might affect the optical properties of the morpholinochlorins. The torsion angles between the two C—C bonds in the morpholine units  in the two morpholinochlorins 1b and 1d vary slightly, with this angle being smaller in the title compound [35.2° in 1b and 25.5 (4)° 1d]. This angle is important as it strongly affects the λmax of the morpholinochlorins .The close structural relationship between the 2,3-disubstituted derivatives 1d in the crystal. Dominant are C—H⋯F hydrogen-bond-like interactions, involving both methyl as well as pyrrole moieties as the hydrogen-atom donor. Weak C—H⋯F interactions involving the solvent are also present. The most prominent of these interactions are given in Table 1Interactions involving fluorine atoms play a dominant role in facilitating the arrangement of molecules of et al., 2019a). Both methoxy substituents are engaged in several of these interactions: C45 exhibits interactions with fluorine atoms from three different pentafluorophenyl groups: with meta fluorine atoms F12iii and F19iv , and one intramolecular interaction with F20, an ortho-fluorine atom. Angular and H⋯F distance values for this intramolecular interaction appear quite unfavorable: the C—H⋯F angle is only 103°, and the H⋯F distance is 2.82 Å. However, only a slight rotation of the methyl H atoms is required to create a much more favorable geometry, and the C⋯F distance between C45 and F20 is at 3.184 (3) quite short . Interactions involving the methoxy group of C46 involve F10v and F1vi, two ortho-fluorine atoms . Two C—H⋯F interactions originate from pyrrole moieties, involving H atoms at the pyrrole moieties flanking the morpholine unit: H8 towards F18i, and H18 towards F8ii, with both F8 and F18 being para-fluorine atoms . These two interactions work in tandem with each other and with a severely slipped π–π stacking interaction, between the rings of F6–F10 and F16i–F20i, connecting two opposite ends of the morpholinochlorin molecule with its neighbors to create infinite chains connected via C—H⋯F and slipped π–π stacking interactions . The centroid-to-centroid distance of the π-stacking interaction is 4.3551 (15) Å, with a ring slippage of 2.795 Å and a centroid-to-mean-plane distance of 3.1661 (12) Å. The last C—H⋯F interaction involves the methylene group of the minor moiety solvate methylene chloride molecule. Given the degree of disorder of the solvate molecules (see Refinement section), this interaction is probably vaguely defined at best and will not be discussed in detail.The most prominent C—H⋯F interactions , interactions between two fluorine atoms are different and much weaker in nature  Å (F5⋯F7vii) and 2.828 (3) Å (F5⋯F8vii) . With the intramolecular distance between F7 and F8 being 2.726 (2) Å, this leads to the formation of a nearly equilateral triangle of F atoms . It should be noted that atom F8 of this F3-triangle also acts as the acceptor of the C18—H18⋯F8ii contact and the backside of the aromatic ring of F8 is involved in the slipped π–π stacking interaction (see discussion above). Fluorine atom F11 features a close contact with a symmetry-created copy of itself, created by a twofold axis. The F⋯Fiii distance here is 2.783 (3) Å, and the C—F⋯Fiii angle is 125.5 (2)° .Besides C—H⋯F interactions, which are generally considered as directional interactions similar in strength to the better investigated C—H⋯O interactions, ms Fig. 6a. It shns Fig. 7a. The ler Fig. 6b. The Fvi and C42vi) of another pentafluorophenyl ring [symmetry code: (vi) −x + y + z + 1]. The F⋯C distances are 3.034 (3) and 2.978 (3) Å for F2 and F3, respectively. There are two interactions of this kind per molecule, one as the C—F donor and one as the π-density moiety accepting the C—F⋯π bond, connecting molecules into centrosymmetric dimers. One of the methyl C—H⋯F contacts (towards F1) is also involved in the formation of these dimers .Besides F11, F2 and F3 are also involved in intermolecular C—F⋯π interactions, pointing nearly perpendicularly towards C atoms , fifteen morpholinochlorins, two thiomorpholines . Disorder with hexane, the other type of solvent used during crystallization, was excluded as a possibility due to the limited size of the solvate pocket, and it is thus assumed that 12% of void spaces in the crystal structure remained unoccupied during the crystallization process.The solvate methylene chloride molecule is disordered over four positions around an inversion center . The C—Cl and Cl⋯Cl distances were restrained to target values and 2 and CH3 moieties, respectively. Methyl CH3 groups were allowed to rotate but not to tip to best fit the experimental electron density. Uiso(H) values were set to a multiple of Ueq(C) with 1.5 for CH3, and 1.2 for CH and CH2 units, respectively.N-bound H atoms were located in a difference electron-density map and were freely refined. H atoms attached to carbon atoms were positioned geometrically and constrained to ride on their parent atoms. C—H bond distances were constrained to 0.95 Å for pyrrole CH moieties, and to 1.00, 0.99 and 0.98 Å for aliphatic CH, CH10.1107/S2056989020009093/is5543sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989020009093/is5543Isup2.hklStructure factors: contains datablock(s) I. DOI: 2013871CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Phormidium ambiguum and Microcystis aeruginosa) were exposed to diurnal light-intensity variation to investigate their favorable and stressed phases during a single day. The photosynthetically active radiation (PAR) started at 0 µmol·m−2·s−1 (06:00 h), increased by ~25 µmol·m−2·s−1 or ~50 µmol·m−2·s−1 every 30 min, peaking at 300 µmol·m−2·s−1 or 600 µmol·m−2·s−1 (12:00 h), and then decreased to 0 µmol·m−2·s−1 (by 18:00 h). The H2O2 and antioxidant activities were paralleled to light intensity. Higher H2O2 and antioxidant levels , and superoxidase dismutase) were observed at 600 µmol·m−2·s−1 rather than at 300 µmol·m−2·s−1. Changes in antioxidant levels under each light condition differed between the species. Significant correlations were observed between antioxidant activities and H2O2 contents for both species, except for the CAT activity of P. ambiguum at 300 µmol·m−2·s−1. Under each of the conditions, both species responded proportionately to oxidative stress. Even under maximum light intensities (300 µmol·m−2·s−1 or 600 µmol·m−2·s−1 PAR intensity), neither species was stressed. Studies using extended exposure durations are warranted to better understand the growth performance and long-term physiological responses of both species.Two harmful cyanobacteria species ( The growth and spread of cyanobacteria have increased, thus threatening today′s water bodies and supplies worldwide ,2. GlobaDuring cyanobacteria control efforts, chemical control measures are discouraged due to their potentially harmful secondary effects on ecosystems ,13,14, wBiological, chemical, and physical factors collectively determine the occurrence and distribution of cyanobacteria in the environment ,25,26. PThe photosynthetic species produce reactive oxygen species (ROS) as a byproduct of the photosynthesis process, which is harmful when accumulated in cells . Therefo2O2) and antioxidant (guaiacol peroxidase (GPX), catalase (CAT), ascorbic peroxidase (APX), and superoxidase dismutase (SOD)) responses of cyanobacteria to diurnal changes in the light intensity were studied. Two photosynthetically active radiation (PAR) levels, 300 and 600 µmol·m−2·s−1, were selected as maximum light intensities, and the responses of two cyanobacterial species, Phormidium ambiguum and Microcystis aeruginosa, were tested for gradually varying light conditions. P. ambiguum is a non-heterocystous filamentous cyanobacterial species, categorized as a benthic cyanobacterium [M. aeruginosa is a floating (buoyant) and colony foaming type cyanobacterium [In this study, the oxidative stress  and M. aeruginosa (strain NIES 111) were obtained from the National Institute for Environmental Studies, Tsukuba, Japan. Both species were cultured for 14 days at 20 °C under a 12 h:12 h light:dark cycle inside an incubator . Light was provided with cool white fluorescent lamps and the intensity was maintained at 20–30 µmol·m−2·s−1 PAR. The nutrient medium was 100% BG-11 [0% BG-11 . During P. ambiguum and M. aeruginosa cyanobacteria cultures were made, maintaining the 0.6 ± 0.02 optical density measured at 730 nm (OD730) using a spectrophotometer . The dilution of the cyanobacteria culture was accomplished with BG11 nutrient medium. In all experiments, the temperature was maintained at 20 °C in an incubator, whereas the lighting conditions changed from 0 µmol·m−2·s−1 (at 06:00 h) to 300 µmol·m−2·s−1 or 600 µmol·m−2·s−1 (at 12:00 h) by changing the lighting intensity by ~25 µmol·m−2·s−1 or ~50 µmol·m−2·s−1 every 30 min with a VBP-L24-C2 light . The light intensity was then decreased at the same rate (until 18:00 h). The lighting condition was controlled with warm light-emitting diode panel lights, and the light intensity was measured using a quantum flux meter . Cyanobacteria samples from each flask were collected for analysis every 3 h, at 06:00 h, 09:00 h, 12:00 h, 15:00 h, 18:00 h, and 21:00 h. To facilitate mixing, each flask was manually shaken at the time of sampling.Following the 14-day incubation, 3 replicate conical flasks  from each of the 2O2 contents were estimated according to standard methods [g for 10 min at 4 °C. The cell pellets were washed once with ultrapure water . To extract cellular H2O2, cell pellets were homogenized in 1 mL of 0.1 M pH 6.5 phosphate buffer and centrifuged at 10,000× g for 10 min at 4 °C. A total of 750 μL of 1% titanium chloride in 20% H2SO4 (v/v) was then added to initiate the reaction. The optical absorption was measured at 410 nm using a spectrophotometer (UVmini-1240), following centrifugation  at room temperature (25 ± 2 °C). The H2O2 concentration was determined using a standard curve, prepared using a series of samples with known H2O2 concentration.Cellular H methods . Brieflyg at 4 °C for 10 min and removing the supernatant and cell pellets, which were homogenized in 1 mL potassium phosphate buffer . A total of 65 µl of enzyme extract was then mixed with 920 μL of potassium phosphate buffer  containing 20 mM guaiacol. With the addition of 15 μL of 0.6% H2O2, the reaction was then started, and the absorbance change was recorded at 470 nm every 10 s for 3 min using UV mini-1240. GPX activity was calculated using an extinction coefficient of 26.6 mM/cm.The GPX activity was assayed as described by Hoda et al.  and MacAg at 4 °C for 10 min. The supernatant was removed, and the cell pellets were homogenized in 1 mL potassium phosphate buffer , containing 0.1 mM EDTA. After centrifuging again , the supernatant was collected as the enzyme extract. The CAT activity was measured by reacting 15 µL of 750 mM H2O2, 920 µL of potassium phosphate buffer, and 65 µL of extract supernatant. Optical absorption was measured at 240 nm using UV mini-1240. The measurements were recorded every 10 s for 3 min, and the CAT activity was calculated using an extinction coefficient of 39.4 mM/cm.CAT activity was measured using the method described by Aebi . A total2O2. Calculations were performed using a molar extinction coefficient for ascorbate of 2.8 mM/cm.APX activity was assayed, as described by Nakano and Asada . The decSOD activities were determined by performing nitro blue tetrazolium (NBT) assays, as described by Ewing and Janero . Each saP. ambiguum and M. aeruginosa were evaluated using a Student’s t-test, assuming equality of variance. Pearson’s correlation analysis was used to evaluate correlations between parameters. Statistical analyses were performed by using IBM SPSS Statistics for Windows, Version 25.0. .One-way analysis of variance (ANOVA), followed by Tukey’s post-hoc test, was performed to test the statistical significance of variations among the means of sample groups. Data were normalized relative to the starting group (06:00 h), by dividing the results of each group by the corresponding 06:00 h group for each replicate. Significant differences between experimental groups of 2O2 contents of P. ambiguum and M. aeruginosa increased with increasing light intensity and peaked between 12:00 h and 15:00 h. The H2O2 content decreased thereafter in parallel with decreasing light intensity. The cyanobacteria were exposed to dark at 18:00 h; however, even at 21:00 h, the H2O2 contents did not reach the initial level measured at 06:00 h . ANOVA testing of P. ambiguum exposed to 600 µmol·m−2·s−1 max PAR intensity grouped H2O2 contents at 06:00 h, 09:00 h, and 18:00 h, and 12:00 h, 15:00 h, and 21:00 h . For M. aeruginosa exposed to 300 µmol·m−2·s−1 max PAR intensity, ANOVA testing grouped H2O2 contents at 06:00 h, 09:00 h, and 21:00 h, 12:00 h and 15:00 h, and 18:00 h . ANOVA testing of M. aeruginosa exposed to 600 µmol·m−2·s−1 max PAR intensity grouped H2O2 contents at each time point . Comparing the H2O2 contents of 300 µmol·m−2·s−1 and 600 µmol·m−2·s−1, max PAR intensity groups showed that the H2O2 contents of both P. ambiguum and M. aeruginosa differed significantly at each time point (p < 0.01 for each time point).The H 06:00 h . For P. P. ambiguum and M. aeruginosa increased over time and reached the maximum at 12:00 h when the maximum PAR intensity was reached (300 µmol·m−2·s−1 or 600 µmol·m−2·s−1). With decreasing light intensity, the GPX activities of both species were decreased. However, with the 300 µmol·m−2·s−1 max PAR intensity group, at 18:00 h and 21:00 h, the GPX activities decreased even further than the starting GPX activity (06:00 h). With the 600 µmol·m−2·s−1 max PAR-intensity, the GPX activity of both species decreased with decreasing light, but the GPX activity of P. ambiguum remained higher than the starting GPX activity, even at 21:00 h. For M. aeruginosa, GPX activity of 600 µmol·m−2·s−1 max PAR intensity reached the starting GPX activity (06:00 h) at 21:00 h . ANOVA testing of the P. ambiguum exposed to 600 µmol·m−2·s−1 max PAR intensity grouped GPX activities at 06:00 h and 09:00 h; 12:00 h and 15:00 h; 18:00 h; and 21:00 h . For M. aeruginosa exposed to 300 µmol·m−2·s−1 max PAR intensity, ANOVA testing groped GPX activities at 06:00 h, 18:00 h, and 21:00 h; 12:00 h; and 09:00 h and 15:00 h . ANOVA testing of M. aeruginosa exposed to 600 µmol·m−2·s−1 max PAR intensity grouped GPX activities at 06:00 h and 21:00 h; 06:00 h, 09:00 h, and 18:00 h; 09:00 h, 15:00 h, and 18:00 h; and 12:00 h . A comparison between 300 µmol·m−2·s−1 and 600 µmol·m−2·s−1 max PAR intensity groups showed that the GPX activities of P. ambiguum and M. aeruginosa differed significantly at each time point (p < 0.01 for each time point).The GPX activities of both  21:00 h . For P. P. ambiguum and M. aeruginosa increased over time but showed a delayed response, as the maximum CAT activities were reached at 15:00 h (which was 3 h after the maximum light intensities of 300 µmol·m−2·s−1 or 600 µmol·m−2·s−1 PAR was reached). Decreasing light intensities were paralleled by reduced CAT activities, although the CAT activity did not reach the initial CAT level, even at 21:00 h . ANOVA testing of P. ambiguum exposed to 600 µmol·m−2·s−1 max PAR intensity grouped CAT activities at 06:00 h and 21:00 h; 09:00 h, 12:00 h, and 18:00 h; and 15:00 h . For M. aeruginosa exposed to 300 µmol·m−2·s−1 max PAR intensity, ANOVA testing groped CAT activities at 06:00 h and 21:00 h; 9:00 h; 21:00 h, 09:00 h, and 18:00 h; and 12:00 h and 15:00 h . ANOVA testing of M. aeruginosa exposed to 600 µmol·m−2·s−1 max PAR intensity grouped CAT activities at 06:00 h and 21:00 h; 09:00 h and 18:00 h; 12:00 h; and 15:00 h . Comparisons between the 300 µmol·m−2·s−1 and 600 µmol·m−2·s−1 max PAR intensity groups showed that the CAT activities of P. ambiguum differed significantly at 09:00 h, 12:00 h, 15:00 h, and 18:00 h (p < 0.01), although the CAT activities at 06:00 h and 21:00 h were not different (p > 0.05). The CAT activities of 300 µmol·m−2·s−1 and 600 µmol·m−2·s−1 max PAR intensity groups of M. aeruginosa also differed at 09:00 h, 15:00 h, and 21:00 h (p < 0.05).The CAT activities of both  21:00 h . For P. P. ambiguum and M. aeruginosa increased with increasing light intensity, but a delayed response was observed, where the maximum APX activity was reached at 15:00 h (3 h after the maximum light intensities were reached). With subsequent decreasing light intensity, the APX activities of both species decreased and reached the initial APX activity (06:00 h) at 21:00 h . ANOVA testing of P. ambiguum exposed 600 µmol·m−2·s−1 max PAR intensity grouped APX activities at 06:00 h, 18:00 h, and 21:00 h; 09:00 h, 18:00 h, and 21:00 h; and 12:00 h and 15:00 h . For M. aeruginosa exposed to 300 µmol·m−2·s−1 max PAR intensity, ANOVA testing grouped APX activities at 06:00 h, 18:00 h, and 21:00 h; 06:00 h, 09:00 h, and 15:00 h; and 12:00 h . ANOVA testing of M. aeruginosa exposed to 600 µmol·m−2·s−1 max PAR intensity grouped APX activities at 06:00 h and 21:00 h; 06:00 h, 09:00 h, and 18:00 h; 09:00 h, 15:00 h, and 18:00 h; and 12:00 h . Comparisons between the 300 µmol·m−2·s−1 and 600 µmol·m−2·s−1 max PAR intensity groups showed that the differences in APX activities for both species were significantly higher in the 300 µmol·m−2·s−1 max PAR intensity groups, from 09:00 h to 18:00 h (p < 0.01 for each light condition for both species). The 21:00 APX activities of P. ambiguum were significantly lower in the 300 µmol·m−2·s−1 max PAR intensity group than in the 600 µmol·m−2·s−1 max PAR intensity group (p < 0.01), although no differences were observed for M. aeruginosa.The APX activities of both  21:00 h . For P. P. ambiguum and M. aeruginosa increased with the light intensity. With decreasing light, SOD activity decreased for both species and approached the starting level at 21:00 h, for both 300 µmol·m−2·s−1 and 600 µmol·m−2·s−1 max PAR conditions . ANOVA testing of P. ambiguum exposed to 600 µmol·m−2·s−1 max PAR intensity grouped SOD activities at 06:00 h, 18:00 h, and 21:00 h; 09:00 h, 18:00 h, and 21:00 h; 12:00 h; and 15:00 h . For M. aeruginosa exposed to 300 µmol·m−2·s−1 max PAR intensity, ANOVA testing grouped SOD activities at 06:00 h and 21:00 h; 06:00 h and 09:00 h; 09:00 h, 15:00 h, and 18:00 h; and 12:00 h, 15:00 h, and 18:00 h . ANOVA testing of M. aeruginosa exposed to 600 µmol·m−2·s−1 max PAR intensity grouped SOD activities at 06:00 h and 21:00 h; 06:00 h, 09:00 h, 15:00 h, and 18:00 h; and 09:00 h and 12:00 h . Comparisons between the 300 µmol·m−2·s−1 and 600 µmol·m−2·s−1 max PAR intensity groups indicated that the SOD activities of P. ambiguum differed at 12:00 h and 15:00 h (p < 0.01) and that M. aeruginosa showed no significant differences between 300 µmol·m−2·s−1 and 600 µmol·m−2·s−1 max PAR intensity conditions (p > 0.05).The SOD activities of both nditions . For P. P. ambiguum and M. aeruginosa increased with the light intensity. With decreasing light, the AOX activity decreased, and, for both species, the AOX activity approached the starting level at 21:00 h, under both the 300 µmol·m−2·s−1 and 600 µmol·m−2·s−1 PAR conditions . ANOVA testing of P. ambiguum exposed to 600 µmol·m−2·s−1 max PAR intensity grouped AOX activities at 06:00 h, 18:00 h, and 21:00 h; 09:00 h, 18:00 h, and 21:00 h; 15:00 h; and 12:00 h . For M. aeruginosa exposed to 300 µmol·m−2·s−1 max PAR intensity, ANOVA testing grouped AOX activities at 06:00 h and 21:00 h; 09:00 h and 18:00 h; and 12:00 h and 15:00 h . ANOVA testing of M. aeruginosa exposed to 600 µmol·m−2·s−1 max PAR intensity grouped AOX activities at 06:00 h and 21:00 h; 06:00 h and 18:00 h; 09:00 h, 15:00 h, and 18:00 h; and 12:00 h and 15:00 h . The AOX activities of 300 and 600 µmol·m−2·s−1 max PAR intensities differed significantly at 12:00 h and 15:00 h, with P. ambiguum (p < 0.01), while those for M. aeruginosa only exhibited a significant difference at 09:00 h (p < 0.01). In other cases, no significant differences were observed.The total antioxidant (AOX) activities of both nditions . For P. 2O2 levels and those of the antioxidants  and the AOX levels were significantly linearly correlated, except for the GPX activity of P. ambiguum under the 300 µmol·m−2·s−1 max PAR intensity , confirming that less variance occurred. When the AOX values were considered, the 2R showed relatively higher values (>0.72).The relationships between the Hntensity . Pearson2O2 contents and the antioxidant activities of P. ambiguum and M. aeruginosa were highly responsive to the diurnal variations in light intensity. In this study, the only variable factor was the light intensity, where H2O2 levels were high during times of higher light intensities and decreased at lower light intensities. When cellular H2O2 level increases, the antioxidant activities correspondingly increase to prevent damage induced by oxidative stress [2O2 levels, the antioxidant activities also varied during the same time frame and followed the H2O2 levels, which increased at higher light intensities and decreased at lower light intensities. The antioxidant activities of both species were correlated linearly with the H2O2 contents. Although the H2O2-antioxidant relationships were varied from strong to weak (depending on the antioxidant species), overall, our findings suggest that the antioxidant levels of both species responded to the cellular H2O2 level accordingly.The He stress ,50. As o2O2 and antioxidant responses followed the same trends for both maximum light intensity conditions (PAR intensities of 300 µmol·m−2·s−1 or 600 µmol·m−2·s−1). Under the maximum PAR intensity of 600 µmol·m−2·s−1, the cyanobacteria received approximately twice the photon energy of the group with a maximum PAR of 300 µmol·m−2·s−1. Therefore, it can be anticipated that the experiment groups, which receive higher photon energy, undergo an enhanced rate of photosynthesis. This is evidenced by the increased H2O2 formed after exposure to a higher light intensity [2O2 production is reduced with higher light exposure [2O2 contents correlated directly with light intensity, even at higher intensities, PAR intensities under 600 µmol·m−2·s−1 did not subject either cyanobacterial species to photo stress. However, this study only involved a single day diurnal variation, and the H2O2 levels of the cells did not reach the starting H2O2 conditions at 06:00 h (even at 21:00 h) for either species. The antioxidant activities almost decreased to the initial conditions by 21:00 h. Therefore, cells may undergo oxidative stress during dark conditions due to the lack of antioxidant activities. If the H2O2 was continued to be presence in cells, the protein synthesis of photosystems will be inhibited [2O2 and adaptation responses.The Hntensity ,52. Howentensity ,54, duriexposure ,56,57. Anhibited  and, in nhibited . TherefoM. aeruginosa than P. ambiguum, except for GPX. Under high H2O2 contents, the AOX activity was highly elevated in P. ambiguum, but in the dark, both species reached the starting AOX activity level at 21:00 h. This finding suggests that M. aeruginosa is less tolerant to oxidative stress than P. ambiguum [2O2 contents, both species demonstrated significant linear relationships (with the only exception being for GPX of P. ambiguum under a maximum PAR of 300 µmol·m−2·s−1). Therefore, despite the high AOX content of the P. ambiguum, both species were able to maintain balanced antioxidant activity under every light condition of the single day exposure.The antioxidant levels differed between the two species, where the response level was lower for ambiguum ,59. ConcM. aeruginosa is a buoyant species that floats in a range of depths and might have higher tolerance to oxidative stress [P. ambiguum. However, in the present study, both species experienced same light intensity variance as the cultures were mixed periodically. The non-different H2O2 contents between the two species suggested that both species experienced similar levels of oxidative stress. Therefore, less oxidative stress tolerance of P. ambiguum triggered the antioxidant activity at a relatively higher rate. Conversely, nonenzymatic antioxidants, primarily carotenoids, protect against ROS in phototrophs, including cyanobacteria [P. ambiguum than M. aeruginosa [M. aeruginosa is due to involvement of nonenzymic antioxidant over the P. ambiguum.The difference in antioxidant levels of the two species can be related to their behavioral characteristics. The e stress ,61 than bacteria ,62. The ruginosa ,64,65; t−2·s−1 and 600 µmol·m−2·s−1) confirmed that the OD730 and chlorophyll-a contents of cyanobacteria  were significantly reduced, which was associated with oxidative stress [Our previous study on the effects of 8 days of exposure to non-varying, high-light intensities (300 µmol·me stress . In futu


Vγ9Vδ2 T cells respond in a TCR-dependent fashion to both microbial and host-derived pyrophosphate compounds . Butyrophilin-3A1 (BTN3A1), a protein structurally related to the B7 family of costimulatory molecules, is necessary but insufficient for this process. We performed radiation hybrid screens to uncover direct TCR ligands and cofactors that potentiate BTN3A1’s P-Ag sensing function. These experiments identified butyrophilin-2A1 (BTN2A1) as essential to Vγ9Vδ2 T cell recognition. BTN2A1 synergised with BTN3A1 in sensitizing P-Ag-exposed cells for Vγ9Vδ2 TCR-mediated responses. Surface plasmon resonance experiments established Vγ9Vδ2 TCRs used germline-encoded Vγ9 regions to directly bind the BTN2A1 CFG-IgV domain surface. Notably, somatically recombined CDR3 loops implicated in P-Ag recognition were uninvolved. Immunoprecipitations demonstrated close cell-surface BTN2A1-BTN3A1 association independent of P-Ag stimulation. Thus, BTN2A1 is a BTN3A1-linked co-factor critical to Vγ9Vδ2 TCR recognition. Furthermore, these results suggest a composite-ligand model of P-Ag sensing wherein the Vγ9Vδ2 TCR directly interacts with both BTN2A1 and an additional ligand recognized in a CDR3-dependent manner.  •Radiation hybrids identify BTN2A1 as crucial for Vγ9Vδ2 phosphoantigen (P-Ag) sensing•BTN2A1 binds directly to the T cell receptor via germline-encoded regions of Vγ9•Cell-surface BTN2A1 associates directly with BTN3A1 independent of P-Ag stimulation•The Vγ9-BTN2A1 interaction modality suggests an additional CDR3-dependent TCR ligand Karunakaran et al. find that butyrophilin 2A1 (BTN2A1) associates with BTN3A1 on the cell surface and binds directly to germline-encoded regions of the Vγ9 chain of the Vγ9Vδ2 TCR. Thus, BTN2A1 collaborates with BTN3A1 to potentiate Vγ9Vδ2 T cell recognition, playing an essential role in phosphoantigen sensing. Human peripheral blood γδ T cells are dominated from an early age by Vγ9Vδ2 lymphocytes , an innaVγ9Vδ2-mediated P-Ag sensing requires cell-cell contact  and depeTursiops truncatus) and alpaca (Vicugna pacos)  . Of notea pacos) . In conta pacos) . Consista pacos) . Furthera pacos)  and canca pacos) . From thOur previous studies have established that BTN3A1 is necessary but not sufficient for P-Ag sensing and indicated the existence of an additional putative Chr-6-encoded factor that synergized with BTN3A1 to stimulate P-Ag-mediated responses , which wWe showed previously that a T cell hybridoma expressing the Vγ9Vδ2 MOP TCR produced interleukin (IL)-2 in co-culture with BTN3A1-transduced Chinese hamster ovary (CHO) cells incubated with the anti-BTN3A1 monoclonal antibody (mAb) 20.1 but exhibited a complete lack of response to HMBPP or Zol . In contTo identify Factor X, we used an unbiased genome-based approach involving generation of radiation hybrids between CHO-Chr 6 cells and BTN3A1-transduced hypoxanthine-aminopterin-thymidine (HAT)-sensitive rodent fusion partners and subsequent analysis of their capacity to stimulate P-Ag sensing by Vγ9Vδ2 T cells A. We posWe fused CHO-Chr6 cells separately with two HAT-sensitive fusion partners—first BTN3A1-transduced A23 hamster cells and second BTN3A1-transduced mouse BW cells  Sanders; resultiHFE, the BTN3A1 gene already transduced into rodent fusion partners, BTN3A2, BTN3A3, and BTN2A1 and BTN2A2. Since we knew that expression of all three BTN3 genes was insufficient for reconstitution of the P-Ag response , BTN2A1 and BTN2A2, which to date have been discussed mainly for their immunomodulatory properties  or BTN2A1 (BTN2A1−/−) or BTN2A2 alone (BTN2A2−/−). Inactivation of both BTN2 genes completely abolished interferon (IFN)γ production by polyclonal Vγ9Vδ2 T cell lines in response to Zol pulsed cells (BTN2−/− and BTN2A1−/− exhibited a complete loss of IL-2 production by TCR-MOP cells in response to either HMBPP (BTN2A2−/− were similar to wild-type (WT) 293T cells  and 49.9 μM (n = 8), respectively . Injection of BTN2A1 IgV produced substantially enhanced signals over surfaces with immobilized Vγ9Vδ2 TCR relative to Vγ4Vδ5 and Vγ2Vδ1 TCRs or control streptavidin surfaces, indicating specific binding A. Equiliectively B.Figure + T cell subset (d 46.6μM [n = 8]) of the BTN2A1-Vγ9Vδ1 TCR interaction  (d 39–50 μM [n = 3]).An initial homology model suggested strong feasibility of a similar interaction mode and highlighted seven amino acids on the face of the BTN2A1 IgV domain incorporating the C, C', F and G β strands (CFG face), equivalent to the region of BTNL3 IgV domain involved in binding Vγ4, as candidates for alanine mutation C. Indivi0–15 μM) D and S3B0–15 μM) . Based o0–15 μM) D and a B0–15 μM) E in the 0–15 μM) F and S3G0–15 μM) G and S3H+ TCR binding  and assessed effects on Vγ9Vδ2-mediated P-Ag response. Although permissive for cell-surface BTN2A1 expression  and CDR2  loops of the Vγ9 IgV domain in BTN2A1 interaction, regions also critical for BTNL3-Vγ4 interaction  Figure F. ConsisCollectively, these findings established that Vγ9Vδ2 TCR binds BTN2A1 IgV via a binding mode that closely mimics that of Vγ4 TCR for BTNL3 and that this binding is essential for P-Ag sensing but occurs alongside parallel and essential Vδ2 CDR-mediated binding events.BTN and BTNL molecules have been shown to form either homo- or heterodimers . To inveInterestingly, the BTN2A1 IgV-C model also highlighted close proximity of extracellular membrane-proximal cysteine residue (C247) with its equivalent residue in the opposing monomer B, suggesFinally, by combining our HADDOCK-derived model of Vγ9Vδ2/BTN2A1 interaction B with ouTo assess whether BTN2A1 and BTN3A1 were associated with each other at the cell surface either before or after P-Ag exposure, a membrane-impermeable amine-reactive cross-linker incorporating a 16Å spacer was used to cross-link proteins on the surface of CHO transductants co-expressing C-terminally HA-tagged BTN2A1 (BTN2A1-HA) and a N-terminally FLAG-tagged BTN3A1 (FLAG-BTN3A1). Immunoprecipitation (IP) using anti-HA beads or 20.1 mAb and subsequent anti-FLAG western blot (WB) was used to detect cross-linked BTN2A1-BTN3A1 species .Figure 5Following IP of BTN2A1-HA using anti-HA beads and subsequent WB detection of FLAG-BTN3A1 under reducing conditions using an anti-FLAG antibody, two discrete bands were detected that considerably exceeded the size of either BTN2A1 or BTN3A1 monomers  A; each w1H-15N-labeled BTN3A1 IgV in E. coli and performed 1H-15N heteronuclear single quantum coherence (HSQC) spectroscopy in the absence and presence of an equimolar amount of naturally labeled E. coli-expressed BTN2A1 IgV . Reagents generated in this study are available on request from the Lead Contact with a completed Materials Transfer Agreement.Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Benjamin E. Willcox  were cultured with RPMI (GIBCO) supplemented with 10% FCS, 1 mM sodium pyruvate, 2.05 mM glutamine, 0.1 mM nonessential amino acids, 50 μM β-mercaptoethanol, penicillin (100 U/mL) and streptomycin (100 U/mL). Peripheral blood mononuclear cells isolated from healthy volunteers were also maintained as above with or without rhIL-2 (Novartis Pharma). 293T cells were maintained in DMEM (GIBCO) supplemented with 10% FCS.6 or 107 cells) were irradiated at Faxitron CP160 . The irradiated cells and fusion partner (BW or A23) were mixed at 1:1 or 1:3 ratio (irradiated cell:fusion partner) and centrifuged at 461 g for 5 min at RT. The cell pellet was gently tapped and 1 mL PEG1500 was added slowly over a minute with gentle mixing in a prewarmed water-bath. After addition of PEG, cells were resuspended in 50 mL warm serum free RPMI and incubated for 30 min, followed by centrifugation at 461 g for 5 min and careful resuspension in RPMI supplemented with 10% FCS at 104 cells/mL. The cell suspension was seeded in 96 well plate flat bottom (A23 fused) or round bottom (BW fused) plates in 100 μl per well. On the following day, 100 μl of 2X HAT was added and cells were selected for two weeks. The selected clones were supplemented with HT medium and further seeded at limiting dilutions to obtain single cell clones which were tested for P-Ag mediated activation of our Vγ9Vδ2 TCR (MOP) transductants. P-Ag presentation capable and incapable clones were PCR characterized for human Chr 6 regions with primers listed in CHO Chr 6  using STAR and the corresponding Gene Transfer Format (gtf) file (version 87). Unmapped reads and those exhibiting more than two mismatches were selected and mapped against the Chinese hamster genome . The corresponding gtf-file was downloaded from the pre-Ensembl ftp site (Cricetulus_griseus.CriGri_1.pre.gtf). Afterward, all unmapped reads and those containing more than two mismatches were again selected to finally map against the human genome . Only reads showing maximally one mismatch were considered as true. With the help of featureCounts, mapped reads were assigned to genomic features using the above mentioned gtf-files. The results were summarized within an Excel-file. Further gene information were extracted from BioMart  and total RNA was extracted. Sequencing libraries were produced with an Illumina Truseq RNA preparation kit as described by the supplier’s protocol and were sequenced with an Illumina HiSeq4000. Sequence reads were mapped to the human genome (hg38) with STAR (version STAR_2.50a) and read counts of gene transcripts were determined using gtf file Homo_sapiens.GRCH38.84.gtf and featureCount (v1.5.0-p1). Cell lines were then compared for presence, i.e., expression, of human Chr 6 genes. To filter out reads descending from mouse or hamster cells, all fastq-files were initially mapped against the mouse genome  as per manufacturer’s protocol.For 6 cells/mL density in a 96 well U bottom plate for 10 days with 100 μL per well. After 10 days, cells were pooled and washed twice and cultivated to rest without rhIL- 2 at 106 cells/mL density in a 6-well plate. After three days, rested cells were subjected for further experiments.Fresh peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteers after obtained written informed consent in accordance with the Declaration of Helsinki and approval by the University of Würzburg institutional review board. Whole blood was layered over the Histopaque-1077 in a 50 mL falcon tube and centrifuged at 400 g for 30 min at room temperature (RT) with no acceleration and brakes. After centrifugation, the opaque interface containing PBMCs were aspirated and washed twice at 461 g for 5 min at RT. Vγ9Vδ2 T cells were expanded by cultivation of PBMCs with RPMI containing 10% FACS, 1 μM BrHPP and recombinant human IL-2 100 IU/mL (Novartis Pharma) in 10BTN2A1 and BTN2A2 genes were disrupted in 293T cells using CRISPR. The CRISPR sequencing targeting functional BTN2A genes were designed with the help of online tools mentioned in the table (software section) and sequences were cloned into GeneArt CRISPR Nuclease vector as per manufacturer’s instructions. On day1, 1.5 × 106 293T cells were seeded in a 6 cm cell culture plate with DMEM medium without pyruvate (10% FCS). On day 2, cells were transfected with 5 μg of BTN2A-IgV_CRISPR cloned GeneArt CRISPR Nuclease (CD4 enrichment) Vector or BTN2A1_49FCRISPR cloned GeneArt CRISPR Nuclease (OFP Reporter) Vector or BTN2A2_343CRISPR cloned GeneArt CRISPR Nuclease (CD4 enrichment) Vector in a calcium-phosphate dependent method  BTN2A1 IgV CRISPR target site2A1IgV       GCAGTGTTTGTGTATAAAGGTGGCAGAGAGAGAACAGAGGAGCAGATGGAGGAGT  552A1allele1  GCAGTGTTT——————————-GTGGCAGAGAGAGAACAGAGGAGCAGATGGAGGAGT  452A1allele2  GCA————————————————-GTGGCAGAGAGAGAACAGAGGAGCAGATGGAGGAGT  39∗∗∗                    ∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗              BTN2A2 IgV CRISPR target site1b) 2A2IgV       GCAGTGTTTGTGTATAAGGGTGGGAGAGAGAGAACAGAGGAGCAGATGGAGGAGT  552A2allele1  GCAGTGTTTGTGTATA-GGGTGGGAGAGAGAGAACAGAGGAGCAGATGGAGGAGT  542A2allele2  GCAGTGTTTG——————————TGGGAGAGAGAGAACAGAGGAGCAGATGGAGGAGT  45∗∗∗∗∗∗∗∗∗∗           ∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗              BTN2A1−/− allelic phenotype2) 2A1-49F      GGACTAGGCTCTAAGCCCCTCATTTCAATGAGGGGCCATGAA-GACGGGGGCATCCGGC  59Allele1      GGACTAGGCTCTAAGCCCCTCATTTCAATGAGGGGCCATGAAGGACGGGGGCATCCGGC  59Allele2      GGACTAGGCTCTAAGCCCCTCATTTCAATGAGGGG——————————————GGCATCCGGC  45∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗                 ∗∗∗∗∗∗∗∗∗∗              BTN2A2−/− allelic phenotype3) 2A2          CAAGAAGGCAGGTCCTACGATGAGGCCATCCTACGCC-TCGTGGTGGCA  48Allele      CAAGAAGGCAGGTCCTACGATGAGGCCATCCTACGCCCTCGTGGTGGCA  49∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗             BTN2−/− cells were seeded overnight in triplicates at 2 × 104 cells/well in 100 μL DMEM containing 10% FCS in a 96 well flat bottom cell culture plate with or without 25 μM Zol. Next day, DMEM with/without Zol was aspirated and cells were washed twice with PBS at RT and 2 × 104 expanded Vγ9Vδ2 T cells/well in 100μL RPMI was added and cocultured for 4 h. After 4 h, supernatants were collected and frozen at −20°C, until cytokine assay was performed with human IFNγ ELISA kit (Invitrogen) was used according to the manufacturer’s instructions.293T or 293T BTN2A1 and BTN2A2 were amplified from the cDNA obtained from 293T cells with the help of pMIM-BTN2A1/2Fwd and pMIM-BTN2A1/2Rev primers , Vδ2-R51A and Vδ2-CDR3 deletion mutant (ΔCDR3: CDR3δ sequence CACD——–YTDKLIF) TCR chains were generated as reported earlier . Vγ9-E70Drosophila S2 cells and purified by nickel chromatography as previously described (cDNA encoding wild type BTN2A1 (S27 to V142) or BTN2A2 IgV domains (S31 to V146), or BTN2A1 IgV incorporating the described mutations, were generated as gblocks (Integrated DNA Technologies) including the sequence for a C-terminal 6x Histidine tag and cloned into the pET23a expression vector (Novagen). Proteins were overexpressed, purified and refolded as described . BTN2A1 escribed . TCRs we5 cells at 4°C.Flow cytometry staining of the samples were performed with the below mentioned antibodies and samples were measured on FACSCalibur or LSRII flow cytometer (BD). The expression of BTN3A1 and BTN2A1 were detected with anti-huBTN3 (CD277) clone 103.2 (gift from David Olive) and anti-huBTN2A1 clone 1C7D (MBL), followed by secondary antibody F(ab’) Donkey anti mouse IgG (H+L) R-PE (Jackson Immunoresearch). mIgG1,κ isotype clone p3.6.2.81 (eBiosciences) and mIgG2a,κ isotype clone-eBM2a (eBiosciences) were used a isotype controls and were detected by above mentioned secondary antibody. N-terminal HA-tagged BTN2A1 or BTN2A2 were detected using anti-HA-FITC (Biolegend). PBMC expanded human Vγ9Vδ2 T cells were detected with FITC- conjugated anti-human Vδ2 (BD Biosciences). Biotinylated soluble Vγ9Vδ2 TCRs were tetramerized by the addition of Streptavidin-PE conjugate (ThermoFisher Scientific) at room temperature, and 1-2μg of tetramer used to stain 10SPR was performed as previously described  on a BIABTN2A1 and BTN2A2 loci have been functionally inactivated (293T BTN2−/− cells), or CHO CD80+ cells, were transduced to overexpress HA-tagged BTN2A1 or BTN2A2, or BTN3A1, as indicated. Cells were surface biotinylated using EZ-Link Sulfo NHS-LC-biotin  for 30 min on ice, quenched with 20mM Tris pH 7.5 for 5 min, washed in TBS, and lysed in lysis buffer containing 1% NP40 in 20mM Tris pH 7.5, 150mM NaCl ± 10mM iodoacetamide (Sigma). HA-tagged BTN2A1 or BTN2A2 was immunoprecipitated using anti-HA resin (BioLegend). Immunoprecipitations were washed in lysis buffer and eluted in nonreducing (NR) or reducing (R) SDS sample buffer and boiled, or incubated at 37C for 5 min before separation on 4%–20% SDS-PAGE gels (BioRad). Proteins were transferred to PVDF using the BioRad TransBlot Turbo system, blocked in 3% BSA, then incubated with streptavidin-HRP (Thermo). To investigate potential association of BTN2A1 and BTN3A1 at the cell surface, CHO cells overexpressing BTN2A1-HA, FLAG-BTN3A1, or both, were treated with the soluble, membrane-impermeable crosslinker sulfo-EGS (ThermoFisher) at 0.5mM in PBS, at 4°C for 2 h. Following this, the reaction was quenched by addition of Tris pH 7.5 to 20mM. Cells were washed in TBS and lysed in 1% NP40 lysis buffer. After centrifugation to remove insoluble material, immunoprecipitation was carried out using anti-HA resin or 20.1 antibody bound to protein A Sepharose . Immunoprecipitates were run on duplicate 4%–20% gels (BioRad) and blotted with anti-BTN2A1 or anti-FLAG antibodies.293T cells in which the The ectodomain structures of BTN2A1 (residues Q29-A248) and BTN2A2 (residues Q33-M265), were generated using the I-TASSER (Iterative Threading ASSEmbly Refinement) server . BrieflyThe BTN2A1-IgV/Vγ9 complex was modeled with HADDOCK . BTN2A1 R65 (located in the IgV domain of BTN2A1) forms a salt bridge interaction with E76 (in the Vγ9 TCR chain). This interaction is likely to be abolished by introducing Ala at this position in BTN2A1 IgV domain (R65A), consistent with abrogation of binding by the R65A mutant. The hydroxyl group of S72 (BTN2A1) is in close proximity to V58 (TCR). Juxtaposition of this polar residue (S72) with a hydrophobic residue (V58) is likely to be energetically unfavorable for binding in this region. By substituting an Ala (ie a non-polar residue) at this position, the S72A mutation is likely to introduce hydrophobic interactions with V58, consistent with enhanced binding compared to wild-type (11-15μM (S72A) versus 50μM (Wild-type)).K79A leads to reduced binding to TCR (100μM). K79 forms a salt bridge interaction with E76 (TCR). Change to Ala will result in loss of this interaction consistent with a reduction in binding affinity. The fact that binding is not totally abolished suggests that this interaction is a not a major contributor to the binding energy. Note however that E76 also contacts R65 (see above).R124A mutation in BTN2A1 abolishes binding to TCR. The HADDOCK model suggests that R124 forms a salt bridge interaction with E70 (Vγ9-IgV TCR) and a hydrogen bonding interaction with the hydroxyl group of T83. These interactions will be lost when introducing an Ala at this position.Y126A abolishes binding to TCR. Y126 forms multiple hydrophobic stacking interactions with I74 (HV4 region of Vg9-IgV). In addition, the hydroxyl group of Y126 forms a hydrogen bonding interaction with T77. These will be lost upon alanine substitution.Although Y133A is located at the interface with Vγ9, it does not mediate interactions with Vγ9 TCR residues and hence it is unsurprising that substitution to alanine does not affect binding affinity. E135 forms a salt bridge interaction with R20 (TCR). Substitution to Ala will result in a loss of this interaction, and consistent with this, E135A mutation abolishes binding to the TCR.1H-15N-labeled BTN3A1. Experiments were processed using Topspin 3.2 (Bruker). All analysis was performed using CCPN Analysis . SPR data was analyzed in BIAevaluation  and Origin 2015 (OriginLab).Stimulation data and transcript visualization in https://doi.org/10.17632/ny6bxn4y9s.1The RNaseq data of radiation hybrid clones filtered for transcribed human genes are available at Mendeley data


However, in the context of human breast cancer, there is a lack of information for potential phenotypic alterations of this crucial immune cell subset. This is partly due to Vδ2+ γδ T cells scarcity in surgical specimens. To break this deadlock, we assessed Vδ2+ γδ T cell phenotypes using untreated breast cancer patients’ peripheral blood, so-called minimally invasive “liquid biopsy”. We show that circulating Vδ2+ γδ T cell phenotypic alterations are already established at diagnosis and related to tumor progression. Notably, terminally differentiated Vδ2+ γδ T cells expressing canonical markers of replicative senescence and exhaustion were significantly associated with tumor-draining lymph node invasion. Our results shed light on the interest of using liquid biopsy to monitor rare events and support Vδ2+ γδ T cell involvement in breast cancer pathogenesis and progression.Vδ2+ γδ T cells in breast cancer is strongly supported by in vitro and murine preclinical investigations, characterizing them as potent breast tumor cell killers and source of Th1-related cytokines, backing cytotoxic αβ T cells. Nonetheless, insights regarding Vδ2+ γδ T cell phenotypic alterations in human breast cancers are still lacking. This paucity of information is partly due to the challenging scarcity of these cells in surgical specimens. αβ T cell phenotypic alterations occurring in the tumor bed are detectable in the periphery and correlate with adverse clinical outcomes. Thus, we sought to determine through an exploratory study whether Vδ2+ γδ T cells phenotypic changes can be detected within breast cancer patients’ peripheral blood, along with association with tumor progression. By using mass cytometry, we quantified 130 immune variables from untreated breast cancer patients’ peripheral blood. Supervised analyses and dimensionality reduction algorithms evidenced circulating Vδ2+ γδ T cell phenotypic alterations already established at diagnosis. Foremost, terminally differentiated Vδ2+ γδ T cells displaying phenotypes of exhausted senescent T cells associated with lymph node involvement. Thereby, our results support Vδ2+ γδ T cells implication in breast cancer pathogenesis and progression, besides shedding light on liquid biopsies to monitor surrogate markers of tumor-infiltrating Vδ2+ γδ T cell antitumor activity.The rationale for therapeutic targeting of Vδ2 Breast cancer (BC) remains the most diagnosed and leading cause of cancer death among women worldwide . AlongsiHowever, exploring antitumor immunity patterns in the tumor bed using resected or biopsied primary or metastatic tumors shows multiple constraints. First, apart from being invasive, tumor biopsies are not always repeatedly feasible and are therefore poorly suitable for longitudinal studies or transfer in clinical routine. Second, genetic and immunological heterogeneity exists between and within each metastasis and the primary tumor, which contributes to the complexity of antitumor immunity pattern study using solid biopsies ,16. Thos+ T cells, γδ T cells display pro-inflammatory cytokine production and cytotoxic effector function. As for αβ CD8+ T cells, γδ T cells can be divided into four functionally distinct subsets reflecting their maturation stages, using a combination of canonical markers known to be differentially expressed during the course of T cell maturation. That is, following antigenic stimulation, CD45RA+ CCR7+ CD27+ CD28+ naïve γδ T cells mature to CD45RA− CCR7+ central memory (CM) γδ T cells with low effector function and strong proliferative potential. Still, upon antigenic stimulation, CM γδ T cells can further mature to CD45RA− CCR7− CD27+/− CD28+/− effector memory (EM) γδ T cells, producing pro-inflammatory cytokines  and cytolytic protein . EM γδ T cells finally mature to CD45RA+ CCR7− CD27− CD28− re-expressing CD45RA terminally differentiated effector memory (TEMRA) γδ T cells with low proliferative potential and strong cytotoxic function [Human γδ T cells represent a unique conserved lineage of T cells, which are responsive against viral/microbial pathogens and transformed cells . Similarfunction ,31,32. W+ γδ T cells since they are the predominant subtype of γδ T cells in breast tissues. Yet Vδ1+ γδ T cell functions remain controversial, either affiliated with a Th1 or regulatory polarization [+ γδ T cells are a potent pro-inflammatory mediator and cytotoxic effectors towards breast tumor cells and have been observed in direct contact with the latter in breast tumors [+ γδ T cells are the major subtype of γδ T cells in the peripheral blood [+ γδ T cells [+ γδ T cells phenotypic alterations in human breast cancers and their impact on disease progression is needed.Contrasting from their counterpart αβ T cells, γδ T cells display a major histocompatibility complex (MHC)-unrestricted antigen presentation and TCR activation, driving their maturation and the expression of natural killer cells (NK) associated cytotoxic receptors. Indeed, γδ T cells also differ from αβ T cells by expressing natural killer cell receptors such as the NKG2 receptors family. Hence, γδ T cells carry hallmarks of innate as well as adaptive immune responses . In brearization ,38,39. Ot tumors ,41,42. Wal blood . Of note T cells ,44,45. H+ γδ T cells phenotypic alteration detectable at diagnosis of early-stage breast cancer. Notably, peripheral PD-1+ or CD57+ EMRA Vδ2+ γδ T cells are associated with the pathological involvement of tumor-draining axillary lymph nodes.To this end, we used mass cytometry to quantify up to 130 immune variables from the peripheral blood of untreated breast cancer patients. We evidenced peripheral Vδ2A total of 122 immune variables were quantified from peripheral blood mononuclear cells (PBMCs) of 13 newly diagnosed BC patients and four healthy volunteers (HV) using two mass cytometry panels Table S1Because there are only two groups, BGA output is a single dimension discriminating axis, where each sample is positioned according to the coexpression of the 122 variables used as input . Still, in the HV group, T cell populations displayed poorly differentiated and non-activated profiles. Indeed, the frequency of Naïve (CD45RA+ CCR7+ CD27+ CD28+) Vδ2+ γδ T cells, Naïve αβCD8+ T cells, Naïve αβCD4+ conventional T cells, central memory  Vδ2+ γδ T cells, resting (CD45RA+ CCR7+ CTLA-4low ICOSlow) Tregs and Tregs expressing a marker associated with a disrupted immunosuppressive activity (DNAM1+), were increased in HV samples compared to BC samples. On the opposite, T cells displaying a differentiated and polarized phenotype were associated with the BC group. Indeed, the frequency of effector memory T cells re-expressing CD45RA  Vδ2+ γδ T cells, TEMRA αβCD8+, late effector memory  αβCD8+ T cells and early effector memory  αβCD4+ conventional T cells were increased in BC samples compared to HV samples. Additionally, an increased frequency of highly cytotoxic (CD8+) NK cells was also detected in BC samples. Importantly, frequencies of Vδ2+ γδ T, αβCD8+ T and NK cells expressing inhibitory receptors  were also increased in BC samples.In the group of HV, we detected an increased frequency of NK cells expressing NK triggering receptors , 7% (NK NKG2C+), 6.3% (Vδ2+ γδ CD4+), 4.7% (Vδ2+ γδ CM), 4.1% (αβCD8+ Naive), 4% (NK IL7R+), 3.5% (Tregs DNAM1+), 3.1%(Tregs resting) and 3%  of contribution to the discrimination of groups (+ γδ KIR2DL1/DS1+), 6.1% (αβCD8+ KIR2DL1/DS1+), 4.5% (Vδ2+ γδ KIR2DL2/DL3+), 4.2% (αβCD8+ LEM), 4% , 3.7% (αβCD8+ TEMRA), 3.5% (αβCD8+ LAG3+) and 3.2% (NK CD8+) of contribution to the discrimination of groups  and six BC patients harboring invaded tumor-draining lymph nodes (BC N+). Among the 130 immune variables, 32, 31, 20, 19 and 28 variables were, respectively expressed by Vδ2ications A.+) highly cytotoxic (CD56+) Vδ2+ γδ T cells profiles. Of note, TEMRA Vδ2+ γδ T cells were the unique population harboring a significantly increased frequency in BC N+ patients compared to BC N− patients  and a canonical marker of T cell functional replicative senescence (CD57+) tends to increase in some patients harboring lymph node invasion, although the difference was not significant. The frequency of NK cells and αβCD4+ T cells expressing inhibitory immune checkpoint receptors  also tended to increase in some patients harboring lymph node invasion.Interestingly, BC N+ patients displayed fully differentiated (TEMRA), activated (ICOSpatients . However+ γδ TEMRA), 7% (Vδ2+ γδ CD57+), 4.5% , 4.1% (Vδ2+ γδ TIM3+), 3.6% , 3.4% (Vδ2+ γδ KIR2DL1/DS1+), 3% (Vδ2+ γδ ICOS+) and 2.7%  of contribution to the discrimination of groups (+ γδ KIR2DL1/DS1+), 6% (αβCD8+ KIR2DL1/DS1+), 5% (Vδ2+ γδ KIR2DL2/DL3+), 4% (αβCD8+ LEM) and 3% , 3.7% (αβCD8+ TEMRA), 3.5% (αβCD8+ LAG3+) of contribution to the discrimination of groups  , cytotoxic potential , activation state , replicative senescence (CD57), inhibitory signaling and tumor-promoting tolerance , susceptibility to apoptosis (Fas), tumor cell recognition and costimulatory signaling (DNAM-1)  A. Twenty(DNAM-1) . Echoing+ γδ T cell clusters that coexpressed inhibitory checkpoint receptors and T cells replicative senescence markers were found to be associated with lymph node involvement (+ PD-L1+ BTLA+ EEM (cluster 2), TIGIT+ CTLA-4+ PD-1+ EEM (cluster 12), CD16+ CD56+ CD57+ PD-1−/+ TIGIT+/− PD-L1−/+ TEMRA  Vδ2+ γδ T cells were exclusively found in patients with lymph node involvement  approved by the institutional review board (Comité d’Orientation Stratégique (COS), Marseille, France) of the Paoli-Calmettes Institute. Written informed consent was obtained from all patients in concordance with the Declaration of Helsinki. Age-matched healthy subjects were recruited from the Etablissement Français du Sang (EFS Alpes-Méditerranée).Breast cancer patients treated at the Institut Paoli-Calmettes were prospectively enrolled between February 2018 and June 2018. Fresh EDTA-anticoagulated blood samples were obtained from patients at diagnosis (n = 13) and healthy volunteers (N = 4). Blood samples were processed extemporaneously. After analysis of morphological tumor characteristics by pathologists, patients were classified as summarized in ® instrument, Fluidigm), at an acquisition rate of approximately 500 events per second. Following the manufacturer’s instructions, settings were on default.PBMCs were obtained using Ficoll-Paque density-gradient centrifugation. Cells were consecutively centrifuged and incubated with cisplatin 0.1 M to stain dead cells. Aspecific epitope binding was blocked with 0.5 mg/mL human Fc Block . The two mass cytometry panels used are provided in + T cells, αβCD8+ T cells, αβCD4+ conventional T cells, Tregs and NK cells were manually gated according to the gating strategy displayed in TCRγδ2+ γδ T cells were exported using FlowJo V10.6.2. Prior to t-SNE analyses using Cytosplore V2.2.1 [+ γδ T cells for each file. The t-SNE analysis was carried out with the default setting (perplexity of 30 and 1000 iterations), and Vδ2+ γδ T cell subpopulations were automatically defined by cell density gradient. The results of the t-SNE analysis for each panel are provided in Manually gated Vδ2e V2.2.1 , consensp-value < 0.05 was considered as significant.Statistical analyses were generated using GraphPad Prism V5.00. Data are expressed as mean ± standard error of the mean (SEM). Statistical significance between two groups was calculated using the nonparametric Mann–Whitney test. A + γδ T cells, phenotypic alterations were evidenced in newly diagnosed breast cancer patients. PD-1+ or CD57+ EMRA Vδ2+ γδ T cells associated with lymph node invasion. These findings evidenced in the context of an exploratory study require confirmation in a larger cohort. However, they open new perspectives for the development of Vδ2+ γδ T cell immunotherapy in breast cancer.Circulating Vδ2


Scientific Reports 10.1038/s41598-020-62990-0, published online 20 April 2020Correction to: This Article contains errors in Table 1 where two column headings are incorrect.d13C (‰)” and “d15N (‰)”.“should read:δ13C (‰)” and “δ15N (‰)”, respectively.“