However, Ca2+-regulated photoproteins activated with CTZ-v display very low bioluminescence activities that hampers its usage as a substrate of photoprotein bioluminescence. Here, we report the crystal structure of semi-synthetic Ca2+-discharged obelin-v bound with the reaction product determined at 2.1 Å resolution. Comparison of the crystal structure of Ca2+-discharged obelin-v with those of other obelins before and after bioluminescence reaction reveals no considerable changes in the overall structure. However, the drastic changes in CTZ-binding cavity are observed owing to the completely different reaction product, coelenteramine-v (CTM-v). Since CTM-v is certainly the main product of obelin-v bioluminescence and is considered to be a product of the “dark” pathway of dioxetanone intermediate decomposition, it explains the low bioluminescence activity of obelin and apparently of other photoproteins with CTZ-v.Coelenterazine- Some of these systems also involve an accessory protein to store a luciferin and deliver it to the luciferase e.g., coelenterazine-binding protein from sea pansy Renilla, or an antenna protein to alter the color and quantum yield of a bioluminescence reaction via Förster resonance energy transfer (FRET) mechanism such as famous green fluorescent protein (GFP)2.Bioluminescence is a natural phenomenon of cold light emission from the living organisms, brought into effect by the chemical reaction in which the enzyme, luciferase, catalyzes oxidation of the substrate, luciferin, by the molecular oxygen. There are more than 30 different bioluminescence systems known comprising various luciferins and luciferases1. In order to oxidize CTZ and produce light, different marine organisms use different enzymes that fall into two categories: classic luciferases such as Renilla, Gaussia, or Metridia ones3 and Ca2+-regulated photoproteins such as aequorin, obelin, or berovin1. In all these cases of various enzymes using CTZ as a luciferin the chemical mechanism of bioluminescence reaction is believed to be the same, however the bioluminescence protein microenvironment plays an important role in shaping the final outcome of each particular reaction thereby offering a wide range of characteristics, such as quantum yield of the reaction, bioluminescence color and kinetics.One of the most widespread and well-studied luciferins is coelenterazine  demonstrates red-shifted bioluminescence with a peak at 532 and a shoulder at 415 nm while obelin with native CTZ emits light with a maximum at 480 nm and a shoulder at 400 nm15.These endeavors were mainly aimed to shift light emission to the longer wavelength region with a view of increasing the detection sensitivity of CTZ-dependent bioluminescent proteins in deep mammalian tissues at their use as reporters for in vivo assays. Since light has its maximum depth of penetration into tissues in the near-infrared window, the demand for the reporters emitting red light is always high. Among the most promising CTZ analogues is coelenterazine-v affects not only spectral properties of the bioluminescence proteins but also the bioluminescence activity and the reaction quantum yield, with the outcome varying from protein to protein. In the case of Renilla luciferase, for example, the use of CTZ-v significantly increases the peak light intensity, providing at that the total light yield comparable to the one of the native CTZ (73%)12. On the contrary, the light outputs of Ca2+-regulated photoproteins and Oplophorus luciferase with CTZ-v are drastically reduced. Thus, when activated by CTZ-v, aequorin and obelin retain only 3.5% and 1.7%, correspondingly14, of the total light yield of photoprotein activated with unmodified CTZ. Unfortunately, it hinders the usage of CTZ-v analogue as a substrate of the photoprotein bioluminescence.However, it quickly became clear that CTZ-v, the crystal structure of Ca2+-regulated photoprotein obelin bound with 2-hydroperoxy adduct of CTZ-v (obelin-v) at 1.80 Å resolution was recently solved15. This structure provided useful information about coelenterazine-binding cavity of obelin-v before the bioluminescence reaction. Unexpectedly, the structures of obelin-v and obelin bound with native CTZ revealed almost no difference; only minor rearrangement in hydrogen-bond pattern and slightly increased distances between the key active site residues and 2-hydroperoxyCTZ-v were found. Thus, the minor changes in the substrate microenvironment observed in the obelin-v active site were proposed to account for the low efficiency of generating an electronic excited state (ΦS) resulting in a low bioluminescence activity of obelin-v15.To shed light on the reasons behind the low efficiency of photoprotein bioluminescence with CTZ-v behavior as a luciferin of photoprotein bioluminescence, determination of the crystal structure of Ca2+-discharged obelin-v was also required, i.e., the structure of obelin-v after bioluminescence reaction with the product bound within the protein active site. In this paper we present the crystal structure of Ca2+-discharged obelin-v bound with the product of bioluminescence reaction at 2.1 Å resolution as well as time-resolved fluorescence properties of Ca2+-discharged obelin-v.To deepen our understanding of the CTZ-2+-discharged obelin-v contains two protein molecules per asymmetric unit  molecules, six DMSO molecules, and 341 solvent molecules. The residues 1–3 in protein chain A and 1–4 in chain B are not visible in the electron-density maps, as is generally observed for the N-terminal residues in the structures of other Ca2+-regulated photoproteins.The crystal structure of Ca2+-discharged obelin-v structure does not contain bound Ca2+ ions, because citrate is a calcium chelator. This makes Ca2+-discharged obelin-v the second photoprotein spatial structure obtained in this particular conformation, i.e., with the reaction product but without Ca2+ ions, the first one being the Ca2+-discharged W92F obelin structure at 1.96 Å resolution (PDB: 1S36)16.Due to the crystallization conditions Ca2+-binding loop of Ca2+-discharged obelin-v in both protein chains A and B (residues 29–37) there is an electron density which plausibly corresponds to Na+. The ion is coordinated by Asp30, Asn32, Asn34, Lys36 and two water molecules with an average of ~ 2.4 Å separation to oxygen atoms that is typical for coordination of both Ca2+ and Na+17. At the same time, Na+ can be straightforwardly distinguished from Ca2+ by electron density, B factors and better Rfree values during refinement (lower by 0.7% in the case of Na+)17. Noteworthy is that the crystals of Ca2+-discharged W92F obelin were also obtained using sodium citrate and Na+ is also found in the first Ca2+-binding site16.However, in the first CaN-terminus. The RMSD of Cα atoms of chain A vs chain B is only 0.22 Å. The notable distinctions were observed in the loop formed by the residues 122–131 which is a part of the EF-hand Ca2+-binding site III that apparently is due to the contacts between the two chains of the dimer. There is a salt bridge defined between Asp123 of the chain A and Asn162 of the chain B, while there is no contact between Asp123 of the chain B and Asp162 of the chain A. Additionally, the conformation of 122–131 loop could be influenced by the presence of intramolecular interaction between Lys124 and Asp134 in the chain A and its absence in the chain B.The structures of chains A and B are almost identical except for the orientations of some side chains on the surface and labile regions of loops and 2+-discharged conformation of obelin-v is a globular molecule with the radius of ~ 25 Å, formed by the two sets of four helices designated as A–D and E–H in the N- and C-terminal domains, respectively  before the bioluminescence reaction19, and that of Ca2+-discharged conformation of obelin with native CTD after the reaction (PDB: 2F8P)20. For example, RMSD of Cα atoms of obelin-v bound with 2-hydroperoxyCTZ-v  and its various mutants, such as F88Y (PDB: 4N1G), W92F (PDB: 1S36), and Y138F (PDB: 4MRY)22. The electron density distribution in the active site of Ca2+-discharged obelin-v strongly suggests the presence of v-modified coelenteramine (CTM-v) pyrazine, i.e., CTM. Later, CTD was shown to be the main product of the aequorin bioluminescence reaction, however, HPLC analysis revealed the presence of CTM24, indicating the established pathway for CTM generation during bioluminescence.The formation of CTM from CTZ was previously reported by Shimomura in his attempts to identify the light-emitting moiety of natural aequorin using denaturing conditionsv resides in hydrophobic internal cavity of Ca2+-discharged obelin in the same position but in different orientation compared to 2-hydroperoxyCTZ-v of obelin-v  is only 0.51 Å . In Ca2+-discharged W92F obelin the benzyl group of CTD is oriented directly to helix B and pushes it out a bit. At the same time the CTM-v of Ca2+-discharged obelin-v is located deeper inside the active center due to the smaller size and its benzyl group is oriented towards helix E. The similar 3.2 Å shift of the helix B (if measured at Ala46 Cα) is present between active obelin-v (PDB: 7O3U) and Ca2+-discharged obelin-v structures that is also related to the benzyl moiety of 2-hydroperoxyCTZ-v pushing the helix B out. Thus, these changes are certainly conditioned by the different size of the reaction product molecule bound in the active center, since CTM-v is much smaller than 2-hydroperoxyCTZ-v and CTD-v.The RMSD Cα of chain A of Cav containing the corresponding 2-hydroperoxyCTZs, the C-terminus effectively caps the substrate-binding cavity, ensuring a solvent-inaccessible and nonpolar environment of the CTZ derivative19. The inaccessibility of the internal substrate-binding cavity of photoproteins to solvent is additionally guarded by the hydrogen bonds formed by Arg21 located in helix A and C-terminal Pro as well as Asp187 and Phe17826.In both obelin and obelin-2+-discharged obelin-v still retains the above mentioned organization of C-terminus cap. However, Ca2+-discharged obelin-v also has a possible solvent-access opening located between Phe28, Leu29 of helix A, Ile42, Ala46, Ile50 of helix B, and Ser142 of helix F , repositioning of Lys45 and Asp49 and breaking of a hydrogen bond between Lys45 and Ser142 can be detected simultaneously with the solvent-access opening appearance. This might be attributed to the high flexibility of Lys45, which could be positioned in four different orientations (two per each chain) and two of which provide a surface opening in question. It is worth mentioning that this ‘hole’ is commonly observed in the structures of other Ca2+-discharged photoproteins, for example, obelin with both CTD and calcium ions or Y138F obelin 22.After the bioluminescence reaction Cax F Fig. c. Relati2+-discharged obelin-v undergoes drastic readjustment. Amino acid residues facing the CTM-binding cavity of Ca2+-discharged obelin-v generally retain the same position and orientation contrary to obelin-v and Ca2+-discharged W92F structures  .Despite minor overall changes when compared to other obelin structures, the organization of substrate-binding cavity of Caly) Fig. . Howeverv forms a hydrogen bond with His22, which is the bond commonly observed in other photoprotein structures -benzyl substituent of 2-hydroperoxyCTZ-v and another molecule is stabilized by the hydrogen bonds with Tyr138, His64 and Trp114  have been used to measure their steady-state and time-resolved fluorescence  or decay-associated spectra (DAS). The first method aims at counting a number of fluorophores providing transformation of the emission spectrum in a nanosecond scale27. The second approach allows distinguishing spectral distribution of the emitters with different fluorescence lifetimes within experimentally observed total spectrum28.Time-resolved fluorescence decays of Ca2+-discharged obelins, the time-resolved area-normalized emission spectra are shown in Fig. 2+-discharged obelin and three—for Ca2+-discharged obelin-v that indicates the presence of two and four emitters with distinct fluorescence lifetimes, respectively29. The presence of two fluorophores in obelin spectra could not be surprising, since, along with the intense band peaked at 515 nm, which is usually attributed to the anionic form of CTD, there is also a weak shoulder around 415 nm . For Ca2+-discharged obelin, two lifetime components assured satisfactory fitting quality, while obelin-v dataset required four lifetime components.In order to reveal photophysical characteristics of the emitters found, we obtained DAS of both Ca2+-discharged obelin sample has the fluorescence lifetime of about 1.52 ns and the spectrum peaked near 480 nm  with very similar spectral properties peaked at 434–440 nm. This spectral similarity was to be expected, because the positions of TRANES isoemissive points were close to each other  or as a product of CTD anion hydrolysis with imine as an intermediate Fig. . Accordi2+-discharged obelin bound with CTM-v determined at 2.1 Å resolution. Comparison of the crystal structure of Ca2+-discharged obelin-v with that of obelin bound with 2-hydroperoxyCTZ-v or with Ca2+-discharged obelin with CTD reveals no significant changes in the overall structure. However, drastic changes in CTZ-binding cavity are observed due to the completely different reaction product, CTM-v. Time-resolved fluorescence measurements discovered the presence of at least four emitters in Ca2+-discharged obelin-v, which might be attributed to different ionic forms of CTM-v and CTD-v. Since CTM-v is identified as the main product of obelin-v bioluminescence by structural data and can be considered as a product of a “dark” pathway of the decomposition of dioxetanone intermediate, it explains the low bioluminescence activity of obelin-v relative to that of obelin activated with unmodified CTZ. This explanation is also supported by the quantum yields of bioluminescence reaction (ΦBL) of cnidarian and ctenophore Ca2+-regulated photoproteins35. The ΦBL value of aequorin bioluminescence amounts to 0.19, while ΦBL of berovin is more than twice lower (0.083). This difference correlates well with the amount of CTD and CTM isolated from Ca2+-discharged aequorin and berovin after bioluminescence reaction33.In this study, we report the crystal structure of the semi-synthetic Cav were obtained from NanoLight Technology, a division of Prolume Ltd. . Other chemicals, unless otherwise stated, were from Sigma–Aldrich and were the purest grade available.Native CTZ and CTZ-Escherichia coli BL21-Gold (DE3) Codon Plus (RIPL) cells transformed with pET19-OL8 plasmid36 carrying the cDNA encoding wild type obelin from Obelia longissima38 were cultivated with vigorous shaking at 37 °C in LB medium containing ampicillin (200 µg/ml). Protein expression was induced with 0.5 mM IPTG at OD590 0.6–0.8 and the cultivation was continued for another 3 h. Recombinant apo-obelin was extracted from E. coli inclusion bodies by 6 M urea as previously reported39. Apo-obelin was purified on a DEAE-Sepharose Fast Flow column , and then concentrated with the use of 10 kDa Amicon Ultra Centrifugal Filters . The concentration of apo-obelin was determined using the corresponding molar extinction coefficient at 280 nm calculated with the ProtParam tool40.For apophotoprotein production, v in a buffer 5 mM EDTA, 10 mM DTT, 20 mM Tris–HCl pH 7.2 at 4 °C. Active photoproteins were separated from apo-obelin, unbound CTZs, and DTT by ion-exchange chromatography on HiTrap Q HP column  as described elsewhere34. Active photoproteins and apo-obelin were eluted as separate peaks with the linear salt gradient of 1 M NaCl in 5 mM EDTA, 20 mM Tris–HCl pH 7.2. Protein concentration was determined with the Dc Bio-Rad protein assay kit.To produce active photoproteins, apo-obelin was incubated overnight with a twofold molar excess of CTZ or CTZ-2+-discharged obelin-v to be obtained, an active obelin-v sample after ion-exchange chromatography was concentrated to 1.0 mg, using 10 kDa Amicon Ultra Centrifugal Filters  with simultaneous replacement of chromatography buffer to a buffer containing 4 mM CaCl2, 20 mM Tris-HCl pH 7.2 at 4 °C. The Ca2+-discharged protein was then concentrated to ~ 10 mg/mL with the same Amicon Ultra Centrifugal Filters.For Ca2+-discharged obelin-v was a solution of 1.6 M Sodium citrate tribasic dihydrate (SG1 kit). Thereafter, the Ca2+-discharged obelin-v crystals were grown for 3–7 days at 20 °C to the final size of 100 μm. For X-ray diffraction analysis, the crystals were picked up from the crystallization drop using fiber loops and were flash-cooled in liquid nitrogen. Prior to freezing, the crystals were cryoprotected by transfer to a crystallization solution containing 20% vol/vol glycerol and soaking them for several seconds.Protein crystals were obtained by the sitting drop vapor diffusion method in 96-well crystallization plates . For screening initial crystallization conditions, the NT8 crystallization robot  and commercially available crystallization screening kits JCSG, PACT and SG1 (Molecular Dimensions) were used. Each sitting drop contained 200 nl of protein solution and 200 nl of reservoir solution. The best condition for crystallization of Ca2+-discharged obelin-v crystals were collected on beamline ID23-1 using Eiger2_16M detector at the European Synchrotron Radiation Facility (ESRF), France. Native diffraction data were indexed, integrated and scaled in P61 space group using the XDS software41. The initial model was obtained by molecular replacement (MR) pipeline of Autorickshaw42 using the structure of active wild type obelin (PDB: 1EL4)18 as a search model. After that, the model was iteratively refined with PHENIX43 and adjusted manually using Coot44. Visualization and superposition of the molecular structures were performed using PyMOL 2.5.0 . Parameters to detect hydrogen bonds were 3.6 Å for an ideal geometry and 3.2 Å for minimally acceptable geometry, 180° for a hydrogen bond cone, and 63° for the maximal hydrogen bond angle45. RMSD was calculated using Align method of PyMOL 2.5.0.Data from the Ca2+-discharged obelin-v was determined with a final resolution of 2.1 Å and deposited at PDB bank under ID 8A9S  equilibrated with 1 mM CaCl2, 20 mM Tris–HCl pH 7.2.The Ca2+-discharged obelins were measured using a Cary 5000 spectrophotometer . Steady-state fluorescence spectra of the proteins were obtained with a Fluorolog-3 spectrofluorometer . The intensity was collected within the ranges of 380–750 nm and of 412–750 nm under excitation at 373 or 407 nm for Ca2+-discharged obelin and obelin-v, respectively. All fluorescence spectra were corrected for photomultiplier spectral sensitivity, inner filter effect and background signal.The absorption spectra of Ca2+-discharged obelin and obelin-v, respectively. Intensity decays were recorded within the ranges of 380–592 nm and 413–650 nm with a step of 2 nm and 3 nm correspondingly for Ca2+-discharged obelin and obelin-v; time resolution was 0.027 ns/channel.Time-resolved fluorescence decays were obtained using a time-correlated single photon counting (TCSPC) method with DeltaHub module of Fluorolog-3 . A NanoLED N-370 with the peak wavelength at 373 nm and pulse duration of < 1.2 ns and DeltaDiode DD-405L with the peak wavelength at 407 nm and pulse duration of < 70 ps were applied for excitation for Ca27:To obtain time-resolved area-normalized emission spectra (TRANES), the following steps were performed28 with the DAS6 software . Time-resolved fluorescence decay at the wavelength λ was described as a sum of two (for obelin) or four (for obelin-v) exponents:E(t) is the instrument response function, τi is the lifetime and i-component of the lifetime at a wavelength λ. The fit quality was evaluated by its global χ2 value and weighted residuals.(i) In order to recover the fluorescence decay parameters (amplitudes and lifetimes), the sets of decays were deconvoluted from the pulse excitation response and fitted by a multi-exponential function using the global analysis approachτi, and steady-state emission spectrum. The equation used was:Iss(λ) is the steady-state fluorescence intensity at λ.(ii) Time-resolved emission spectra (TRES), plotted as intensity vs wavelength, were constructed using t was equal to the area of the spectrum at t = 0.(iii) Finally, TRANES were constructed by normalizing the area of each TRES so that the area of the spectrum at time λ:Decay-associated spectra (DAS) were obtained as follows:Supplementary Information.


Valvatidae, a group of freshwater operculate snails near the base of Heterobranchia with a nearly worldwide distribution. This update includes corrections and many additions  to the 2014 contribution and adds all newly described species (11) during the past eight years. The extensive reference list is directly linked, where possible, to the available electronic source of the cited papers.This contribution is an update to the 2014 compilation of all supra- and (infra-) specific taxa of extant and fossil  Melanopsidae or Paludomidae. My work inspired and facilitated several recent papers on the taxonomy of Valvatidae. In particular, Russian authors cleared up the status of many uncertain taxa and provided valuable data and high-quality images of type material  is still hampered by the unavailability of key literature. Many important and also quite recent taxonomic papers on fossil taxa have been written in non-Roman alphabets and often are not available as a digitized version. Thanks to the kindness of several colleagues, I could access some of the most important papers in particular by Russian or Chinese authorities, and the results of checking the original descriptions and their circumstances are compiled and reviewed herein.In contrast, the paleontological analysis of Valvatidae generally is still limited. Also, a robust phylogeny of the family is still in its infancy, since only a few papers have provided molecular data on valvatids  is likely to represent a species complex spread all over the Palaearctic and (by introduction) also the Nearctic region.Whereas there is constant progress in detecting and describing new valvatid species, concerning both fossil and extant taxa Table , our undds e.g., . In partAs a result, there have not been any recent proposals of new genera or further revisions to the supraspecific classification. Accordingly, this update is limited to the species level. The present update adds two extant and nine fossil species recently described, two replacement taxon names, another 21 mainly fossil taxa previously overlooked, and nearly 40 names based on confusions, misspellings, or invalid publications : Two Ph.D. theses are discussed, since they contain the description of new taxa of Bohaispiridae and many hydrobiid genera; see Valvatidae they are listed in alphabetic order below and the names are marked as fossil taxa by “†”:A widely cited Chinese-language work, “Youlou 1978” is particularly problematic. Previous authors, myself included, did not realize that this name actually means “Editors” or “Editorial Board” in Chinese, and no individuals are identified as the authors of either this publication or specific sections of this publication. I was uncertain about the true authorship of the very many taxon names of fossil gastropods introduced in this work. Indeed, the authorship “Youluo” appeared in all cases in the online type catalogue of the Nanjing Institute as well as in the Zoological Record, and has been repeatedly cited by later authors. Meanwhile I was able to check the original paper and can confirm the term “Youluo” as the true  authority in all cases [checked by a native Chinese colleague]. Most important, the actual names of any member of this group are not provided anywhere in this volume. However, ICZN Art. 14 clearly states that “A new name or nomenclatural act published after 1950 with anonymous authorship [Art. 50.1] is not thereby made available”. Accordingly, all species names (more than 120 new taxa!) as well as higher taxa  applanata Zhu X., 1995† Liratinabasicarinata”† “Tropidinabellireticulata”† “Liratinafahaniuensis”† “Liratinahedobia”† “Aphanotylushumeratus”† “Valvatamagniumbilicata”† “Costovalvataminuta”† “Valvata (Atropidina) pileiformis”† “Liratinaqikouensis”† “Valvata (Cincinna) rehetaiensis” → Valvatarehetaiensis Zhu G.-X., 1980† “Valvataringentis”† “Liratinatuozhuangensis”† “Valvatazhouqingzhuangensis”† “Details on these names are outlined below under each name.Valvatidae: The page number for the taxon name in my previous work Remarks: Valvataalpestris Küster, 1853 (Nom: 16)Type locality: “in kleinen Seeen an der Quelle des Giessbaches ohnweit des Faulhorns bei Grindelwald” (Küster 1853: 87). According to current maps this is probably the “Schwarzseeli” near the Faulhorn at Grindelwald, Switzerland.Types are figured by Valvataalta K.A. Ali-Zade, 1932 (NEW)† Original source: K.A. Ali-Zade 1932: 21, pl. 2: figs 12–14 .Remarks: Junior homonym of Valvata (Cincinna) altaica Popova & Starobogatov   mentioned in “Remarks: A nomen nudum like many other similar cases in the catalogue of the Godeffroy Museum Original source: Type locality: Département de l Oise, France.Valvatacangshanensis Pan, 1982 (Nom: 28)† Original source: Valvatacarinata Fuchs, 1870” (Nom: 28–29)† “Valvatacarinata Sowerby, 1834. Accordingly, the name has been meanwhile replaced by Muellerpaliapseudovalvatoides Neubauer, Harzhauser, Kroh, Georgopoulou & Mandic, 2014 (Hydrobiidae).Remarks: As stated in Cincinna  chankensis Prozorova, 1988” (NEW)“mentioned in Cincinna  hankensis Prozorova, 1988.Remarks: Misspelling of Cincinnachersonica Chernogorenko & Starobogatov, 1987 (Nom: 29)ZIN 1/41-1974) mentioned by Type material: Valvataunicariniferachiknaformis” (NEW)† “mentioned in Horizon: Upper Cretaceous.Locality: near the villages of Butera and Machhaghoda (Chhindwara District: Madhya Pradesh) ca. 160 km north of Nagpur , eastern Dekkan Plateau, India.Material: “Holotype”: InS1199 (Appendix 2 (Nom: 137), fig. N; 6.8 mm × 7.6 mm), deposited at University of North Dakota, Grand Forks, North Dakota, USA.Remarks: This subspecies name was mentioned in an unpublished PhD thesis (no ISBN or ISSN numbering) and thus is not formally described as required by ICZN Art. 8.1.3 and 8.6.Valvata (Cincinna)” circinata  mentioned in † “Paludinacircinata)Original source: Type locality: Limestone banks von Sornetan and Tramelan, Val de Délemont/Bezirk Delsberg, Kanton Jura, Switzerland.Type horizon: Lower to middle Miocene.Paludinacircinata, a nomen nudum). ICZN Art. 50.1.1. states clearly that “If the identity of that other person is not explicit in the work itself, then the author is deemed to be the person who publishes the work”, making Greppin the author of Paludinacircinata. However, contrary to the classification by Lithoglyphus. It is also not identical to those specimens from a nearby locality, which are illustrated and described by Valvatacircinata . Accordingly, both Paludinacircinata Greppin, 1855 and Valvatacircinata Sandberger, 1871 remain valid taxa.Remarks: Referred to Valvatapiscinalisvar.cistopolitana G.I. Popov” (NEW)† “cistopolitana nov.”, a nomen nudum in cited as “Remarks: I could not find any trace of this name in the papers of G.I. Popov or in subsequent papers, accordingly a “taxon inquirendum”, probably a nomen nudum, not available.Valvata (Cincinna) confusa  (Nom: 31)Remarks: Valvatabicarinatavar.connectans B. Walker, 1906 (NEW)Original source: Type location: Lake Michigan, New Buffalo, Michigan, USA.Type material: No. 24142 of coll. Walker (presumably now in University of Michigan Museum of Zoology). Alan Kabat (pers. comm. 30 Jan 2022) could not find it in the UMMZ database, but the sample may not yet be catalogued online.Neritacontorta Müller, 1774 (Nom: 32)Type material: Valvatacosinensis Stache, 1889 (NEW)† Original source: Type locality: In old coalmines, north of Cosina (today Hrpelje-Kozina) , Slovenia.Stomatopsis horizon, Paleocene  (Amnicolidae).Remarks: Stache himself noted the high similarity with Valvatacupensis” mentioned in “Valvatakupensis Fuchs, 1870.Remarks: misspelling of Aphanotylusdakangensis Pan, 1982 (Nom: 35)† Original source: Valvata? dalaziensis Zhu G.-X., 1980 (Nom: 35)† Original source: Type locality: Yanji, Jilin, Northeast  impressa” (NEW) Mentioned by V.impressa Pfeff.” [Pfeffer?] by http://www.bibliotekar.ru/5-prareki-chetvertichnyi-period/7.htm“Valvataimpressa”. The malacologist Georg Pfeffer (1854–1931) did not describe any Valvata species. The name is likely a double misspelling of Valvatadepressa Pfeiffer, 1821.Remarks: I could not find any description of a “Valvatainconspicua C.B. Adams, 1851 (Nom. 55)Nanivitreainconspicua  (Cochliopidae) (Remarks: currently considered as iopidae) .Borystheniaintermedia Konrashov, 2007 (Nom: 56)† Paleontological Institute, Russian Academy of Sciences (PIN) #5148/1.Type material: Holotype deposited at Borystheniajalpuchense Gozhik, 2002 (Nom: 57)† Type locality: near Vinogradovka village, Odesa oblast, Bolhrads’kyi district, Ukraine.Type horizon: Miocene, Middle Pontian.Type material: Holotype  figured by jalpuchensis, since Borysthenia is feminine. Morphometric data of the shell compared with Borystheniamenkeana  were provided by Remarks: The species name should be Valvatajaponica Martens, 1877 (Nom: 57)Museum für Naturkunde, Berlin  is figured by Type material: 1 syntype (of 2) from the Remarks: Valvatajiaolaiensis Yu, Salvador, Wang, Fang, Neubauer, Li, Zhang & Wan, 2021 (NEW)† Original source: Yu et al. 2021: 5, fig. 3M–P.36°15'55"N, 119°57'04"E), northern part of Jiaozhou City, Shandong Province, China.Type locality. LK-1 borehole † Original source: Type locality: YHS492, Heping township, Jinggu County, Yunnan Province, China.Type horizon: Bajocian/Bathonian fluvial-lacustrine horizon in the Hepingxiang Formation of China, Middle Jurassic (171.6–164.7 Mya).Type material: Holotype deposited at Nanjing Institute of Geology & Paleontology NIGP #24247.Valvata (Cincinna) joncheryacensis Wenz, 1923” (NEW)† “WMSDB).Mentioned in Valvatajoncheryensis Wenz, 1930. Remarks: Misspelling of †Aphanotylusjurassicus Pan, 1980  (Nom: 58)† Original source: Cincinnakamchatica Prozorova & Starobogatov, 1998 (Nom. 58)ZIN 1/97-1911) and 21 paratypes (ZIN 2/97-911) in Zoological Institute of the Russian Academy of Sciences, St. Petersburg (ZIN).Type material: Holotype  karameilica Wei, 1984 (Nom: 58)† Original source: Wei in Xinjiang Dizhi Ju 1984, 84, pl. 49: figs 1, 2.Type locality: Yandi’s booth: Qitai Jubei Yingxun, Xinjang province, China.Type horizon: Quishan Street, Upper Triassic.Valvata (Tropidina) kebapcii Odabasi, Glöer & Yildirim, 2015 (NEW)Original source: 39°31'30.8264"N, 26°17'9.57"E, 81 m altitude.Type locality: Turkey, northwestern Anatolia, Ayvacik town, Tuzla Stream, COMULM-G 0050 .Type material: Holotype Cincinnakizakikoensis Fujita & Habe, 1991 (Nom: 59)Remarks: Valvata  klemmi Schütt, 1962 (Nom: 59).Type material: A paratype (FS/8894) is stored in the Biologiezentrum Linz (Austria) .Valvatafluviatilisvar.kliniensis Milachevich/Milaschewitsch, 1881 (Nom: 59)56°27´14"N, 36°50´54"E.Type locality: According to ZIN), #1 in systematic catalogue.Type material: 56 syntypes in the Zoological Institute of the Russian Academy of Sciences, St. Petersburg  klucharevae Starobogatov, 1985 (Nom: 60)ZIN). The specimen with number ZIN 1/523-2014 is a topotype that is not included in the type series.Type material: There are no type specimens in the collection of the Zoological Institute of the Russian Academy of Sciences † Original source: 40°46'14.74"N, 42°18'21.05"E, WGS84), Oltu-Narman Basin, northeastern Turkey.Type locality: Kömürlü  and paratypes .0019/0016-0019), all from type locality and horizon, are deposited at the Valvata (Cincinna) korotnevi Lindholm, 1909 (Nom: 60)Remarks: Remarks: Valvata (Cincinna) alienavar.korotnewi Ldh. 1909” mentioned in “Valvata (Cincinna) korotnevi Lindholm, 1909, correctly listed by Remarks: Misspelling of Valvatakournasi Glöer & Hirschfelder, 2019 (NEW)Original source: Type locality: Nómos Chaniá, outflow of Lake Kournas, 3 km southeast of Georgioúpoli, Crete, Greece.ZMH 140040: also figured by ZMH 140041) are stored in the Zoological Museum of Hamburg, Germany (ZMH), remaining paratypes (9 + 1 juv.) in coll. Hirschfelder .Type material: Holotype (Remarks: The anatomy of this species is unknown because only subrecent (fossil or dead) shells have been found.Megalovalvatakozhovi Sitnikova, 1983 (Nom: 60)Remarks: Valvatavancianavar.kubanica Krestovnikov, 1931 (NEW)† Valvatakubanica Krestovnikov, 1929Original source: Type locality: Sediments of Estuary of Velykyi Kuyalnik River near Odessa, northwest Black Sea, Ukraine.Type horizon: Pleistocene.Valvatakurensis” mentioned in “Valvatakupensis Fuchs, 1870.Remarks: Misspelling of Valvata  laethmophila Bekman & Starobogatov, 1975 (Nom: 61)Remarks: Valvatalanta” mentioned in “Valvatalauta Lindholm, 1909.Remarks: Misspelling of Valvata (Cincinna) andreanavar.latior Menzel, 1904 (Nom: 62)† Type horizon: Quaternary.BGR) starting with # BGR-B-ORIG-000181564 to # BGR-B-ORIG-000181888.Type material (more than 20 specimens without designation as types) is stored in the Geozentrum Hannover ZIN 1/601-1986) and 10 paratypes (ZIN 2/601-1986 and ZIN 3/601-1986) in the Zoological Institute of the Russian Academy of Sciences, St. Petersburg (ZIN). The type series includes the holotype and 10 paratypes. Holotype figured by Type material: Holotype , with the middle of the lake forming the border between the Ninglang County of Yunnan Province and the Yanyuan County of Sichuan Province, China.Valvata sp. from Lake Lugu”, resp. “clade CN07/1”. According to their analysis the species belongs to the subgenus Tropidina.Remarks: Not described or figured by the authors or subsequently, thus a nomen nudum. According to Amplovalvatamagna Pan, 1980  (Nom: 65)† Original source: Yu and Pan 1980: 150, pl. 2: figs 5, 6.Valvatamagniumbilicata” mentioned in † “Remarks: Not available from there (see above). I could not find a subsequent reference on this species that would validate the name.Provalvatamaior Cataldo, Lazo, Luci & Aguirre-Urreta, 2019 (NEW)† Original source: Type locality: Quebrada del Gastrópodo, Mendoza Province, Argentina.Type horizon: La Tosca Member, Huitrín Formation, facies D1, Barremian, Lower Cretaceous.MCNAM-PI) 24524.3; paratypes MCNAM-PI 24524.1, 24524.5, and 24524.6; Colección de Paleontología, Universidad de Buenos Aires, Ciudad Autónoma de Buenos Aires, Argentina (CPBA) 23706.1.Type material: Holotype deposited in Museo de Ciencias Naturales y Antropológicas Juan Cornelio Moyano, Colección de Paleontología de Invertebrados, Mendoza, Argentina † Original source: Valvatamathiasi Esu & Girotti, 2018 (NEW)† Original source: Esu & Girotti, 2018: 49–54, figs 2, 3.Type locality: Sambuca Lago Piccolo, Tavarnelle Val di Pesa, Tuscany, Italy.Type horizon: Lower Pliocene.SMF) 349126, from SLP3;Type material. Holotype Senckenberg Museum Frankfurt , Roma, Italy, MPUR7-3959/50, 3960/80, 4153/20.paratypes Valvata known.Remarks: the largest true Valvata (Cincinna) mengyinensis (Grabau)” mentioned in † “Bithyniamengyinensis Grabau, 1923, which is shown in the same figure as Valvatasuturensis by Bithyniamengyinensis), fig. 7e–g ).Remarks: probably an error for Valvatamicroscopica Nevill, 1889 (Nom: 68)Cyclostremamicroscopica, which was reproduced by Type material: According to microscopica  .Remarks: Cincinna  sibiricamiddendorffi Starobogatov & Zatravkin, 1985 (Nom: 69)Remarks: Valvataminima Hislop, 1859  (Nom: 69)† Type horizon: Upper Cretaceous (not Tertiary).NHMUK); the original Latin description was translated to English.Type material: Lectotype designated and figured by Valvataminima Fuchs, 1877” (Nom: 69)† “Valvataminima Hislop, 1859. Accordingly, the name was replaced by Pseudamnicolawelterschultesi Neubauer, Harzhauser, Kroh, Georgopoulou & Mandic, 2014 (Hydrobiidae: Pseudamniocolinae). Valvataminima Fuchs, 1877 with Valvataserbica Brusina, 1902 (an available name).Remarks: As outlined this name is a junior homonym of Provalvataminor Cataldo, Lazo, Luci & Aguirre-Urreta, 2019 (NEW)† Provalvata sp. in = Original source: Type locality: Quebrada del Gastrópodo, Mendoza Province, Argentina.Type horizon: La Tosca Member, Huitrín Formation, facies D1, Barremian, Lower Cretaceous.MCNAM-PI) 24523.3, paratypes MCNAM-PI 24523.2, 24523.4 and 24523.5; Colección de Paleontología, Universidad de Buenos Aires, Ciudad Autónoma de Buenos Aires, Argentina (CPBA) 23704.1.Type material: Holotype deposited in Museo de Ciencias Naturales y Antropológicas Juan Cornelio Moyano, Colección de Paleontología de Invertebrados, Mendoza, Argentina † “Original source: Type locality: Qingjiang Basin, Jiangxi, China.Type horizon: Linjang Formation, Eocene.Type material: Nanjing Institute of Paleontology and Geology, sample # 93414.Valvataminutissima Wattebled, 1884 (Nom: 70).MNHN-IM-2000-33594).Type material: Clenchiellapapuensis Benthem Jutting, 1963 and provided details on morphology, SEM photographs of the operculum , radula , anatomy (fig. 15), and COI sequences (fig. 33) of this species, which is now treated as Clenchiellaminutissima  .Remarks: Valvatamontanaensis Meek, 1876 (Nom: 71)† Remarks: In a published abstract of a conference, Valvatamontenegrina Glöer & Pešić, 2008 (Nom: 71)V.montenegrina is figured by MZ027632 and MZ027633.Remarks: The holotype of Valvatamontenegrinus Glöer & Pešić, 2008” in “Valvatamontenegrina Glöer & Pešić, 2008 (no reason given for gender change).Remarks: Misspelling of Valvatamulticarinata Hislop, 1859  (Nom: 73)† Type horizon: Upper Cretaceous (not Tertiary).NHMUK); the original Latin description was translated to English.Type material: Lectotype designated and figured by Valvatanowshahrensis Glöer & Pešić, 2012 (Nom: 75)Holotype also figured by Valvatanikosi” mentioned in Esu and Girotti (2015: 78) (NEW)† “Pyrgulanikosi Esu & Girotti, 2015 and Valvatapyramidula Esu & Girotti, 2015, both described in the same paper.Remarks: Mismatch of Cyclostomaobtusum Draparnaud, 1801 (Nom: 76)Type material  have been located by Valvatapiscinalis O.F. Müller, 1774.Remarks: Several Russian authors regard this species as valid, whereas most European authors consider this to be a synonym of Valvataoctonaria Brusina, 1902 (Nom: 76)† Valvatasimplex Fuchs, 1870 (non Gould 1847) by Muellerpaliaoctonaria  (Hydrobiidae).Remarks: The taxon was ranked as subspecies of Valvataoecsensis Soós, 1934” mentioned in † “öcsensis” in the cited publications was erroneously emended to “oecsensis”. However, the correct emendation following ICZN rules is “ocsensis”, since it is not derived from a German word (ICZN 32.5.2.1). Current status: Valvataocsensis Soós, 1934.Remarks: Valvata  olkhonica Bekman & Starobogatov, 1975 (Nom: 77)Remarks: Valvatalewisivar.ontariensis Baker, 1931 (Nom: 77)Valvatalewisi Currier, 1868. The authors also provided good photographs of the shells of both taxa.Remarks: Recently, Valvataoregonensis” mentioned in † “V.whitei and V.calli] in 1910 by Hannibal”). Since the two names previously published are both available, the name Valvataoregonensis is not valid.Remarks: As already outlined by Valvatacristatapalustris Kormos” twice mentioned in GNI (Nom. 79)“Valvatacristata Müller, 1774 and Lymnaeapalustris Müller, 1774, both listed in Remarks: As assumed in Valvata (Cincinna) pamirensis Starobogatov, 1972 (Nom: 79)ZIN 1/241-1955) and paratype (ZIN 10/241-1955) from the type locality  in the Zoological Institute of the Russian Academy of Sciences, St. Petersburg (ZIN).Type material: Type data were provided by Valvatapanagilae” [sic! not panagile] mentioned in † “V.catariane, V.olgae, V.mariae, V.theocleti) from Pliocene – Pleistocene sediments of the Ptolemaida Basin  are all extensively described and figured by Remarks: This and four other fossil species  lautavar.parvula Kozhov, 1936 (Nom: 79)Remarks: Cincinnapenglaizhenensis Pan, 1982 (Nom: 81)† Original source: Valvata (Atropidina) pileiformis” mentioned in † “Remarks: Not available from there (see above) or from Valvata (Liratina) baicalensisvar.piligera Lindholm, 1909 (Nom: 83)Megalovalvatapiligerapiligera]. nudicarinata (fig. 6F) and minor (fig. 8D).Remarks: Neritapiscinalis O.F. Müller, 1774 (Nom: 83)Type material: Valvata  platispira Szőts, 1953 (NEW)† Original source: Szőts, 1953: 33, 145, pl. 2: figs 13–15.Type locality: “Hosszúharasztos” (Harasztos quarry), Gánt, District Fejér, Hungary.Type horizon: Eocene.Valvata (Cincinna) polita A.A. Ali-Zade, 1973 (NEW)† Original Source: A.A. Ali-Zade 1973: 155–156, pl. 45: figs 1a, 1b.Type locality: Bozdag (hill), Lower Absheron Peninsula of Azerbaijan.Type horizon: Lower Akchagylian, uppermost Pliocene.MES MSU). The holotype has inventory number (MES MSU # 28/308).Type material: Dr Pavel Frolov  provided information that the fossil molluscs described by A.A. Ali-Zade in 1973 are all stored in the Museum of Earth Sciences of Moscow State University  pontica Pană, 1990 (NEW)† Original source: Pană, 1990: 63, pl. 1: figs 3–10.Type locality: Valley Croitorului, village Sibiciul de Jos, district Buzău, Romania.Type horizon: Upper Miocene, lower Pontium.Type material: Holotype No. 674, Collection Laboratoire de Paléontologie III g, Bucarest, Romania.Valvatasimplexvar.polycincta Lörenthey, 1906 (Nom: 85)† V.simplexoctonaria by Vatributarylvatasimplex), the current status is Muellerpaliahaszprunarioctonaria Neubauer, Harzhauser, Kroh, Georgopoulou & Mandic, 2014 (Hydrobiidae).Remarks: The name was synonymized with Valvata  profundicola Bekman & Starobogatov, 1975 (Nom: 87)Remarks: Valvata (Borysthenia) pronaticina Lindholm, 1932 (NEW)† Original source: Type locality: East border of creek Betekei, a tributary of river Ischim near Selim-Dzhevar; district Petropavlovsk, province Akmolinsk (now Astana), North Kazachstan.Type horizon: Lower Pliocene.CNIGR) # 412–419/3355 (see ZIN 1/359-1935).Type material: stored in the Central Scientific Research Geological Exploration Museum named after F.N. Chernyshev .Remarks: probably misspelled and confused with Costovalvatapulchra” mentioned in “Remarks: nomen nudum, not available.Neritapusilla Müller, 1774 (Nom: 88)Original source: Type material: Not mentioned by Valvatidae and not to a neritid.Remarks, The figures (25 and 26)) in the article “Berlinisches Magazin” (an author is not identified) certainly refers to a species of Valvatapygrncea” mentioned at many webpages, e.g., https://commons.wikimedia.org/wiki/File:The_Quarterly_journal_of_the_Geological_Society_of_London_(1867)_(14595069379).jpg (NEW)“Valvatapygmaea C.B. Adams, 1849 because of erroneous text recognition.Remarks: Misspelling of Valvatapyramidula Esu & Girotti, 2015 (NEW)† Original source: Type locality: S-SW of Neos Erineos, Greece .Type horizon: Upper Lower Pleistocene, grey-yellow silty clays of the Synania Formation.SMF 345727, paratypes SMF 345728/3).Type material in Senckenberg Museum Frankfurt (holotype Liratinaqikouensis” mentioned in † “Remarks: Not available from there (see above) or from Valvata (Cincinna) racovetzae Popova & Starobogatov  (NEW)† Original source: Type locality: The Chuya Basin (or Chuya Steppe), southeastern part of the Altai Mountains, Russia.Type horizon: Kyzylgir formation, Middle to Upper Pliocene.ZIN 1/533-1968).Type material: Holotype deposited in the Zoological Institute of the Russian Academy of Sciences, St. Petersburg  rakovetzae” mentioned in † “Valvata (Cincinna) racovetzae: (1) The figure legend at page 23 (first mention in the paper) states “racovetzae”. (2) The same paper includes the description of Anisus (Pseudocarinogyraulus) racovetzae  and Odhneripisidiumracovetzae , showing the general preference of the authors for the latter spelling. (3) The second author, Valvatacf.racovetzae Pop. Et Starob.”. On the other hand, Dr Pavel Kijashko (pers. comm. 22 Feb 2022) stated that “I believe that the name “rakovetzae” is a priority. It is indicated in the original description and on the original identification label by the hand of Starobogatov it is written: “V.rakovetzae, holotype”. Perhaps it is Starobogatov’s typo (it cannot be clarified now), but it does not contradict ICZN.” The matter may be decided by the Commission of Zoological Nomenclature.Remarks: Probably a misspelling of † Valvata (Cineinna) [sic!] rehetaiensis Zhu G.-X., 1980  (Nom: 105)† Original source: Type locality: Mordoy area, east Sibiria , Russia.Type horizon: Lower Cretaceous .Cyclostomatricarinata Say, 1817 (Nom: 105)Remarks: Valvatatuostaiensis Wei, 1984 (Nom: 107)† Original source: Wei in Xinjiang Dizhi Ju 1984, 84, pl. 49: figs 3, 4.Type locality: Wuyiju Tostai, Xinjang Province, China.Type horizon: Taxihe Formation, Miocene.Liratinatuozhuangensis” [not Valvatatuozhuangensis as stated] mentioned in † “Remarks: Not available from there (see above), nor from Valvataturbinata Stache, 1889 (NEW)† Original source: Type locality: Caracaea-oogones, north of Cosina (today Hrpelje-Kozina), (5 km east of Trieste) Slovenia.Stomatopsis horizon, Eocene.Type horizon: Conus-like shell, a very doubtful member of Valvatidae.Remarks: Because of the Valvataturbinoides K.A. Ali-Zade, 1936 (NEW)† Original source: K.A. Type locality: near Naftalan, west Azerbaijan.Type horizon: Akchagylian, uppermost Pliocene and lower Pleistocene.Valvataalta K.A. Ali-Zade, 1932, a junior homonym of Valvataalta Deshayes, 1862 , Bythinellidae). Not treated in the last 50 years.Remarks: Replacement name for Valvataturkmena” mentioned in A.A. “Pyrgulaturkmena A.A. Ali-Zade, 1967, described in the same volume.Remarks: Probably a misattribution of Valvatapiscinalis (Müll.) var.uistopolitana Pop[ov]” (NEW) mentioned in “Valvatacistopolitana (see above).Remarks: A misspelling of Valvataumbilicata Parreyss” is also mentioned in “Valvataunicarinifera Hislop, 1859 (Nom: 108)† Type horizon: Upper Cretaceous (not Tertiary as stated by Hislop).NHMUK); the original Latin description was translated to English.Type material: Lectotype designated and figured by Valvataunicariniferaunicarinifera” mentioned in † “Locality: near Kalmeshwar (station InL0096b), west of Nagpur, Savner Subdivision of Nagpur district in the state of Maharashtra, India.Horizon: Cretaceous.Material: “Holotype”: InS1159 (Appendix 2 (Nom: 137), fig. O; 1.5 mm × 1.7 mm) stored at University of North Dakota, Grand Forks, North Dakota, USA.Valvataunicarinifera Hislop, 1859 by Bingle-Davis in the same work (see above for “V.u.chiknaformis”), the original taxon becomes the nominal subspecies and cannot be replaced. Thus, the specimens of Bingle-Davis are formally not named and remain to be described in accordance with the rules of ICZN.Remarks: Although freely available online, this PhD dissertation lacks ISBN or ISSN numbering, thus is not formally published. Moreover, in erecting a second subspecies of Valvatasimplexvar.unicincta Lörenthey, 1906 (Nom: 109)Valvatasimplex) the parent species name is not available and has been replaced by Muellerpaliaoctonariaunicincta  (Hydrobiidae).Remarks: As outlined above †Original source: Type locality: Kama tributary of the Middle Volga, Russia.Type horizon: Pleistocene, Sokol Suite of the Kinel Formation.Type material: unknown.Remarks: also mentioned by Danukalova and Morozova (2003: 80) and Matoshko et al. (2004: 21).Valvatavaciani Nourn.” mentioned in Yahimovich et al. (2000: 65) (NEW)“Valvatavanciana Tournouër, 1875.Remarks: misspelling of Valvatavenciana” mentioned in “Valvatavanciana Tournouër, 1875.Remarks: misspelling of Cincinna (Cincinna) vinogradovskaense [sic] Gozhik, 2002 (Nom: 110)† Type locality: near Vinogradovka village, Odesa oblast, Bolhrads’kyi district, Ukraine.Type horizon: Miocene, Middle Pontian.Type material: Holotype  figured by vinog radovskaensis, since both Cincinna and Borysthenia are feminine. “Cincinna (Cincinna) vinogradovkensis” mentioned in Borysthenia Lindholm, 1914.Remarks: The species name should be Valvata (Microcincinna) vystitiensis Chernogorenko & Starobogatov, 1987 (Nom: 111)Type material: Type data provided and holotype figured by Valvataheidemariaewillmanni Neubauer, Harzhauser, Kroh, Georgopoulou & Mandic, 2014 (NEW).† Original source: Type locality: Vokasia-Tal, Kos, Greece.Type horizon: Lower Pleistocene, Middle Iraki-Formation.Type material: According to Valvataheidemariaebicarinata Willmann, 1981, a junior homonym of Valvatabicarinata Lea 1841.Remarks: Replacement name for Valvatawindhauseni Parodiz, 1961 (Nom: 112)† Potamolithus Pilsbry, 1896 .Remarks: Valvatayongkangensis Yü, 1980 (Nom: 112)† Original source: Type horizon: Middle Jurassic.Valvatazhongjiangensis Pan, 1982 (Nom: 113)† Original source: Valvatazhouqingzhuangensis” mentioned in † “Remarks: Not available from there (see above) or from


Presynaptic activation of Ca2+-dependent adenylyl cyclases represents an important mechanism of synaptic transmission modulation. This involves activation of the cAMP-PKA pathway to regulate neurotransmitter synthesis, release and storage, and to increase neuroprotection. This aspect is of paramount importance for the preservation of neuronal survival and functionality in several pathological states occurring with progressive neuronal loss. Hence, the aim of this review is to discuss mutual relationships between cAMP and Ca2+ signaling and emphasize those alterations at the Ca2+/cAMP crosstalk that have been identified in neurodegenerative disorders, such as Alzheimer's and Parkinson's disease.It is commonly accepted that the role of astrocytes exceeds far beyond neuronal scaffold and energy supply. Their unique morphological and functional features have recently brough much attention as it became evident that they play a fundamental role in neurotransmission and interact with synapses. Synaptic transmission is a highly orchestrated process, which triggers local and transient elevations in intracellular Ca Nowadays, it is commonly known that astrocytes are highly specialized types of glial cells responsible for synapse formation and regulation of ongoing neuronal transmission. They also interact with other glial cells or blood vessels depending on the brain region  that allow detection and reaction to neuronal signals  are capable of eliciting diverse pleiotropic responses, thus regulating basic cellular functions, such as growth and differentiation, gene transcription and protein expression as well as astrocyte-mediated synaptic plasticity, gliotransmission, energy supply and maintenance of the extracellular environment  as well as genetically encoded sensors for cAMP and protein kinase A (PKA), allowed for high-resolution detection of astrocytic Ca2+/cAMP fluxes in different physiological and pathological conditions  into Gα subunit and βγ dimer by the exchange of GDP for GTP. Based on the sequence homology and functionality of α-subunits, G proteins are divided into four main families: Gsα, Giα/Goα, Gqα/Gα11, and Gα12/Gα13 that regulate distinct downstream signaling events  that leads to hydrolysis of phosphoinositol diphosphate (PIP2) into diacylglycerol (DAG), known as a membrane-bound regulator of cAMP concentration, and inositol 1,4,5-triphosphate (IP3), known as a soluble messenger triggering the release of Ca2+ ions from the endoplasmic reticulum (ER)  but the mechanism of its activation and inhibition by Ca2+ remains largely unexplored and controversial  in astrocytes may be negatively modulated by neuron-derived factors which may alter the Ca2+ response triggered by ionotropic receptors , mitochondrial Ca2+ uniporter (mCU), and mitochondrial permeability transition pore  channel. Activation of this receptor can induce constitutive D-serine release from astrocytes for NMDA receptor-dependent LTP maintenance  and N-methyl-D-aspartate (NMDA) receptors, both gated by glutamate, and ATP-gated purinergic P2X receptors  and plasma membrane Ca2+ ATPase (PMCA) are the main regulators of Ca2+ extrusion to the extracellular space while the control of Ca2+ concentration in the ER is ensured by the sarco/endoplasmic reticulum calcium ATPase (SERCA) , respectively. In astrocytes, cAMP is regulated mainly by the activation of adenosine receptors (A1 and A2A), adrenergic receptors (α2 and β1−3), dopamine receptor (D1/5), glutamatergic receptor (mGlu3), histamine receptor (H2), PACAP/VIP receptors, serotonin receptors (5-HT4 and 5-HT5A) and also opioid receptors, all summarized in 2+ and cAMP signaling. It is assumed that all of the nine tmAC isoforms, which have also been identified in mice astrocytes , CaM kinase (CaMK), calcineurin (CaN), protein kinase C (PKC), or Gq-coupled receptor activation. For instance, AC8 activated in a Ca2+/CaM-dependent manner binds the Orai1 channel, which is a major functional component responsible for store-operated calcium entry , Epac1 and Epac2, or PKA or cAMP-gated ion channels called hyperpolarization-activated cyclic nucleotide-gated (HCN) channels  to maintain the exocytotic secretion of gliotransmitters  as well as antioxidant activities, especially glutathione-related defenses. Among genes downregulated by cAMP stimulation, the overwhelming number of encoded modulators of the physiological process as cell cycle, proliferation, or death , as well as cytoskeletal proteins and mediators of the immune system. Therefore, cAMP is involved in the regulation of a plethora of cellular functions from the antioxidant systems through the control of the normal state of differentiated astrocytic cells  classified into 11 families, some of which are regulated by Ca2+ messenger systems in astrocytes is non-linear and of great complexity. Undoubtedly, spatial and temporal resolution should be taken under consideration to understand their synergistic and antagonistic relationship.It is apparently becoming clear that the existence of an interplay between cAMP and Ca2+ handling. The Aβ-induced astrocytic metabolic failure, including the production of reactive oxygen/nitrogen species, Ca2+-dependent glutathione depletion, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation and mitochondrial Ca2+ dyshomeostasis, is ultimately linked to synaptic dysfunction and neuronal death  and PI3K-Akt signaling pathway in cortical astrocytes cultures  plaques and neurofibrillary tangles composed of hyperphosphorylated protein tau are commonly detected hallmarks of Alzheimer's disease (AD). Neurodegeneration manifested by neuronal integrity loss and white matter lesions or gliosis, is widely recognized in postmortem brain of AD patients , and D2-like receptor family coupled to Gi/o  to stimulate or inhibit AC/cAMP/PKA transduction pathway, respectively. All five subtypes of dopamine receptors are also expressed in astrocytes. In cultured cortical astrocytes, the application of SKF83959 led to discovery of non-cyclase-coupled D1-like receptor called phosphatidyl-inositol-linked D1 receptor which induces Ca2+ mobilization via the Gq/PLC/IP3 signaling , the second most recognized neurodegenerative disorder. The α-synuclein proteins secreted from neurons are easily taken up by astrocytes through endocytosis causing inflammatory response such as release of cytokines , oxidative stress and mitochondrial impairment  defects have been repeatedly demonstrated in numerous neurodegenerative diseases, especially AD, PD but also Huntington's disease . It is believed that this mechanism of intercellular communication may be linked to astrocytic purinergic activation and participation of ATP release to autoinflammation in the CNS  and DA neuronal death generating parkinsonian phenotypes  level in brain tissue of patients with PD , especially type 4 (AQP4). It is highly concentrated in glial endfoot membranes and forms a molecular complex with a transient receptor potential cation channel subfamily V member 4  grant no. 2019/33/B/NZ4/00587 and by the Medical University of Lodz grant no. 503/6-086/-2/503-61-001.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In addition, astrocytes are capable of releasing several biologically active substances, including glutamate and GABA. Astrocyte-mediated GABA release has been a matter of debate because the expression level of the main GABA synthesizing enzyme glutamate decarboxylase is quite low in astrocytes, suggesting that low intracellular GABA concentration ([GABA]i) might be insufficient to support a non-vesicular GABA release. However, recent studies demonstrated that, at least in some regions of the CNS, [GABA]i in astrocytes might reach several millimoles both under physiological and especially pathophysiological conditions, thereby enabling GABA release from astrocytes via GABA-permeable anion channels and/or via GABA transporters operating in reverse mode. In this review, we summarize experimental data supporting both forms of GABA release from astrocytes in health and disease, paying special attention to possible feedback mechanisms that might govern the fine-tuning of astrocytic GABA release and, in turn, the tonic GABAA receptor-mediated inhibition in the CNS.Astrocytes are the most abundant glial cells in the central nervous system (CNS) mediating a variety of homeostatic functions, such as spatial K Such a As astrocytes express aquaporins , their m2+-activated Cl− channels i of 150 nM. This concentration is quite close to the resting [Ca2+]i of astrocytes, suggesting the possibility of tonic GABA release under resting conditions. Pharmacological blockade or genetic silencing of Best1 in cerebellar astrocytes reduces the GABAA receptor (GABAAR)-mediated tonic current in cerebellar granule cells by about 75%. These results indicate that Bergmann glia cells and lamellar astrocytes in the mouse cerebellum tonically release GABA via Best1 channels under resting physiological conditions i in astrocytes, confirming the involvement of the Ca2+-modulated Best1 channel. Thus, similar to cerebellar astrocytes, [GABA]i is quite high in thalamic astrocytes and Best1 channels the mediate constitutive [Ca2+]i dependent release GABA under resting physiological conditions nt study . In contstudy i) increases high enough  strongly depends on the brain region. In the early postnatal cerebral cortex, [GABA]o has been reported to be 0.25 µM i of ~1 mM in the cerebral cortex, ~4 mM in the hippocampus, and ~10 mM in the thalamus  and values available in the literature . Interestingly, the dumping of astrocytic [Ca2+]i transients affected mouse behavior, as evident by potentiated self-grooming. Electrical activity of medium spiny neurons (MSNs), striatal output neurons, was reduced, although neither glutamatergic nor GABAergic phasic synaptic transmission has been modified. The observed reduction in MSN activity was mediated by tonic GABAergic inhibition via extrasynaptic GABAA receptors. Unexpectedly, the blockade of GAT3, but not of GAT1, rescued the strength of tonic GABAAR-mediated inhibition and alleviated the self-grooming behavior. The further experiments demonstrated that the reduction in Ca2+ activity reduced the number of GAT3 inserted in the plasma membrane, while the mere GAT3 expression was unaffected i is required to change the direction of GAT3-mediated GABA transport. Furthermore, [Ca2+]i influences the number of plasma membrane located in GAT3 and in turn the strength of the GAT3-mediated release/uptake.In summary, tonic GAT3-mediated GABA release from astrocytes occurs in the cerebral cortex probably because of low [GABA]Alzheimer´s disease (AD) is a neurodegenerative disorder with an increasing prevalence in aging populations. Several lines of evidence suggest that amyloid β (Aβ) peptide and tau protein aggregations trigger a cascade of pathological changes, including the weakening of synaptic transmission and synapse loss . AstrocAR-mediated inhibition in the dental gyrus in the hippocampus has been reported in the 5xFAD mouse model of AD i by the D-aspartate-triggered stimulation of EAAT switched the GAT3 in the release mode, similar to that in WT animals. Thus, it was suggested that in the healthy striatum, the EAAT-mediated glutamate uptake keeps GAT3 in the reverse mode by means of elevated [Na+]i  i and switch of GAT3 in uptake mode; (2) reduced number of membrane-located GAT3; or (3) reduced [Ca2+]i with decreased Ca2+-dependent Best1 conductance, remains elusive.In summary, the experimental data demonstrate that the GABA release from striatal astrocytes is reduced in HD. However, the question whether the observed reduction results from (1) reduced i increase i elevation can switch the GAT3 into reverse mode  GABA synthesis in hippocampal astrocytes and (2) results in astrocytic GABA release. The question of whether the GABA release is driven by the EAAT-mediated [NaI in the range of 0.5–4 mM and [GABA]o in the range of 0.5–1 µM. For both channel- and/or transporter-mediated GABA releases, experimental evidence has been provided, as summarized in the previous paragraphs and in Theoretical assumptions indicate that, depending on the region and functional state, both the channel- and/or transporter-mediated GABA release is possible, given an astrocytic [GABA]o shifts its reversal towards more positive values, thus favoring the uptake mode ly EAATs , may shiEAATs i levels determine both the threshold and rate of tonic GABA release via this pathway. Interestingly, Bergmann glia cells in the cerebellum demonstrate variable resting [Ca2+]i, from about 50 to 250 nM, and the amplitude of somatic ATP-induced Ca2+ responses is negatively correlated to the resting somatic [Ca2+]i [2+]i is spatially heterogeneous, ranging from 40 to 120 nM, and local [Ca2+]i controls the amplitudes of sensory-induced Ca2+ transients in vivo [2+]i of about 150 nM [2+]i ranging from about 50 to 250 nM are within the dynamic range of Best1, thus supporting the activity-dependent modulation of the Best1-mediated GABA release. However, mechanisms underlying spatially restricted [Ca2+]i levels and thus the focal control of the Best1-mediated GABA release needs further investigations.Best1 is a Ca [Ca2+]i . Similar [Ca2+]i . Moreove in vivo . Becauset 150 nM , the repo. The reversal of GAT directionality in Bergmann glial cells was reported to occur when [GABA]i reaches about 10 mM i of 3 mM o  into the reverse mode. The NCE-mediated Ca2+ influx can stimulate the ATP release from astrocytes, leading to an adenosine-mediated inhibition of glutamate activity [+]i stronger than GATs, one can suggest that the GAT3- and/or EAAT-mediated [Na+]i increase reverse NCE, resulting in a near-membrane [Ca2+]i increase. Such local Ca2+ transients may open Best1 channels, leading to the GABA release  physiological contribution is determined as mainly neuroglial interactions at several levels.Definitely not only changes in astrocytic activity or intracellular homeostasis determine the strength and direction of the GABA transport. In the hippocampus under resting conditions, the extracellular GABA has a mostly neuronal origin , and thease mode . Physiolreviews, ,131. Suc2+ concentration. In addition, the local intracellular Ca2+ concentration determines the Best1 conductance, while the intracellular Na+ concentration influences the GAT3 reversal potential. We suggest that the astrocytes possess both GABA release pathways. Local ionic signaling and GABA gradients, also as a consequence of neuronal activity, may play a decisive role to switch on/off a particular mechanism of GABA release. Elucidating the mechanisms governing GABA release may help to develop therapeutic tools for the treatments of pathophysiological conditions.In this review, we have summarized the experimental results reporting the GABA release under physiological and pathophysiological conditions. Increasing evidence demonstrates that astrocytes are able to synthesize GABA using different metabolic pathways. Intracellular GABA concentration may be as high as 10 mM. Given that the extracellular GABA concentration is in a low micromolar range, the transmembrane GABA gradient enables both Best1-channel- and GAT3-transporter-mediated GABA release. Indeed, the tonic GABA release from astrocytes has been demonstrated in the cortex, cerebellum, thalamus and striatum. Interestingly, in the cerebellum and thalamus, GABA is predominantly released via the Best1 channel, while in the cortex and striatum, the GAT3-mediated GABA release dominates. In diseases, the GABA release from astrocytes is typically facilitated and again, in some cases, the Best1-mediated release is potentiated, whereas, in other cases, the GAT3-mediated pathway plays the dominating role. The question why nature uses one or the other pathway remains elusive. The expression and trafficking of Best1 and GAT3 is modulated by several intracellular factors, including the intracellular GABA and Ca


The tumor microenvironment (TME) plays an essential role in the development, proliferation, and survival of leukemic blasts in acute myeloid leukemia (AML). Within the bone marrow and peripheral blood, various phenotypically and functionally altered cells in the TME provide critical signals to suppress the anti-tumor immune response, allowing tumor cells to evade elimination. Thus, unraveling the complex interplay between AML and its microenvironment may have important clinical implications and are essential to directing the development of novel targeted therapies. This review summarizes recent advancements in our understanding of the AML TME and its ramifications on current immunotherapeutic strategies. We further review the role of natural products in modulating the TME to enhance response to immunotherapy. Acute myeloid leukemia (AML) is a complex hematologic malignancy driven by the abnormal proliferation and differentiation of immature myeloid precursors ,2. The rProgress in deciphering the remarkable complexity and heterogeneity of AML has led to the development of selected therapies for certain AML subsets. Chemotherapy remains the mainstay treatment for AML over the decades. The use of combination of cytarabine and anthracycline, known as the “3+7 regimen”, hematopoietic stem cell transplantation (HSCT), and, more recently, the use of targeted therapies, such as hypomethylating agents , venetoclax, FLT3- inhibitors, and IDH inhibitors, have resulted in promising responses in select AML subsets [The TME comprises a complex network of stromal cells , immune cells , the extracellular matrix (ECM), and secreted factors, such as cytokines  Figure . AlthougMesenchymal stromal cells (MSCs) are non-hematopoietic progenitors that constitute an essential component of the bone marrow (BM) niche and are well known to possess important immunomodulatory function and the ability to regulate the development and differentiation of HSCs via direct cell-to-cell contact and release of a wide array of soluble growth factors and cytokines ,7,8. TheEvidence exists to suggest some degree of T lymphocyte dysfunction in AML . HoweverRegulatory T cells (Tregs) are a CD4+ CD25+ T cell subset critical to maintain the peripheral homeostasis and central tolerance through their ability to suppress the proliferation/function of T helper cells. Over the past decade, studies have observed abnormally high levels of Tregs in the BM and peripheral blood of patients with AML compared with healthy donors ,23. MoreIn recent years, natural killer (NK) cells have been increasingly identified for their role in tumor immune surveillance through direct cytotoxicity, as well as indirect influence on other immune cells in the TME . Using aMyeloid-derived suppressor cells (MDSCs) are a heterogeneous population of CD11b+ CD33+ HLA-DR-immature myeloid cells with immunosuppressive function. The expansion of MDSCs has been well documented in AML patients, are associated with poor outcomes, tumor progression, and are believed to be involved in hampering the efficacy of immune-based therapies ,37,38. HTumor-associated macrophages (TAMs) are a major component of the TME, and the polarization to M2 phenotype in AML has been shown to subvert antitumor immunity and promote tumor progression ,41. ReceThe secretion of soluble environmental factors, such as chemokines, cytokines, and growth factors, play an indispensable role in shaping the immunosuppressive milieu. Although AML blasts may prompt the secretion of pro-inflammatory cytokines, such as tumor necrosis factor-a (TNF-a), IL1b, and IL6, AML blasts also secrete immunoinhibitory factors, such as IL-10, TGF B, IL-35, and indoleamine 2,3-dioxygenase 1 (IDO1) ,44,45. OChemokines orchestrate the migration and adhesion of immune cells within the TME and are known to play a prominent role in AML progression. The expression of the chemokine CXCL12 by mesenchymal stromal cells has gained considerable attention as a potential therapeutic target in AML. CXCL12 and its receptor CXCR4 provide critical signals to prompt cell survival, adhesion, and migration. Furthermore, our lab, as well as others, have demonstrated the importance of the CXCL8-CXCR1/CXCR2 signaling pathways, which has more recently been intertwined with the upregulated expression of macrophage migration inhibitory factor (MIF) ,47,48,49Several soluble metabolic factors have also been cited for their immunosuppressive capabilities in AML. Notably, the expression and release of arginase II by AML blasts has been shown to alter the immune microenvironment by enhancing arginine metabolism. By limiting arginine availability within the TME, AML blasts promote T cell exhaustion and polarize surrounding monocytes toward an suppressive M2-like phenotype ,51. The Alteration of the TME by AML blasts leads to increased resistance, recurrence, and progression of the disease. To better understand and discover potential treatment modalities, scientists have identified a few specific strategies deployed by AML blasts for alteration of the TME. Through the expression of immune checkpoint inhibitors, alteration of the formation of T cell immune synapses, secretion of immunoinhibitory soluble factors that affect T cell responses, and proliferation of myeloid-derived-suppressor cells (MDSC) and macrophages, AML blasts can facilitate T cell dysfunction.NK cell dysfunction is achieved with altered expression of NK-cell ligands, such as MICA, ULBP1, ULBP2, and ULBP3 via epigenetic modifications, thus leading to alteration of the activating receptor NKG2D, which rely on those ligands, and through induction of co-inhibitory receptors in NKs, such as TIGIT, which inhibit IFN-y release. Increased TIGIT expression decreases the amount of NK cells available in marrow and is associated with poor outcomes. In addition to this, the hypoxic and inflammatory environment and metabolic reprogramming all contribute to the stromal and vascular niches in the TME, facilitating progression of the disease. Particularly, mesenchymal stromal cells (MSC) have been shown to inhibit both innate and adaptive immunity in hematological malignancies, however the theory behind how this is achieved is still being reviewed. Of note, recent literature indicates that NK cells are significantly less effective in destroying AML blasts when they are cultured with MSCs, highlighting the protective effect of the stromal microenvironment. This protective effect towards the AML blasts is hypothesized to be mediated via TLR4. Differences in MSC expression of inhibitory and pro/anti-inflammatory ligands have been noted across different clinical/cytogenetical subgroups of AML, further emphasizing the important role that they play in the pro-AML TME and the additional research needed to further elucidate their role in immunomodulation and effect on treatment with immunotherapies.Altered immune cell homing via manipulation of the CXCL12/CXCR4 axis plays a major role in the survival, growth, and chemotherapeutic resistance of AML blasts. This key migratory axis is strongly implicated in leukemic stem cells (LSCs) relocation in bone marrow. Particularly, overexpression of CXCR4 on AML blasts portends poor prognosis, as it facilitates trafficking of malignant LSCs within BM. This effect is further amplified by decreased MSC expression of CXCL12 in AML, which reduces migration of nonmalignant stem cells in BM. Additionally, AML blasts are known to induce the downregulation and/or loss of HLA1 and HLAII leading to defective antigen presentation. Due to the complex interplay within the TME, immunotherapies have begun to be geared towards combinatorial strategies. The bone marrow microenvironment is a critical player in the NK cell response against acute myeloid leukemia in vitro ,56.Altered immune cell homing via manipulation of the CXCL12/CXCR4 axis plays a major role in the survival, growth, and chemotherapeutic resistance of AML blasts. This key migratory axis is strongly implicated in leukemic stem cells (LSCs) relocation in bone marrow. Particularly, overexpression of CXCR4 on AML blasts portends poor prognosis, as it facilitates trafficking of malignant LSCs within BM. This effect is further amplified by decreased MSC expression of CXCL12 in AML, which reduces migration of nonmalignant stem cells in BM. Additionally, AML blasts are known to induce the downregulation and/or loss of HLA1 and HLAII leading to defective antigen presentation. Due to the complex interplay within the TME, immunotherapies have begun to be geared towards combinatorial strategies ,56.Future directions will be focusing at overcoming multiple immunosuppressive mechanisms, as well as at targeting non-malignant components of the TME, such as stromal cells and vascular components, and enhance the immune-related effects.Recognized as one of the hallmarks of cancer, immunomodulation leading to cancer cell evasion of the innate immune system has been researched in detail for the past half-century, leading to the development of novel therapies targeting the immune system and the tumor microenvironment associated with immune evasion. The immune system acts on and shapes malignant cells in a process called “immunoediting” which consists of three recognized steps: elimination, equilibrium, and escape. Elimination involves the innate and adaptive immune responses acting to eradicate developing tumors. Equilibrium, considered to be the most dangerous phase because it is the longest, is the continuous sculpting of tumor cells, which can lead to resistant variants emerging that have increasing resistance to immune pressure. Finally, in the escape phase, those resistant variants overcome the immune pressure and escape from immunomodulation, allowing them to grow uncontrollably. This final phase is where most of the current research and clinical trials are focused. Current immunotherapies try to take advantage of the three major principles of escape mechanisms: alteration of tumor or effector cells leading to the lack of antigenic recognition of malignant cells, resistance to cell death, and induction of immunotolerance and ignorance through alteration of the tumor microenvironment (TME) via secretion of immunosuppressive factors. The TME in AML and the various treatment modalities that are being trialed are the focus of this review ,57.The expression of inhibitory checkpoint (IC) molecules on AML blasts has been recognized as an important mechanism of immune evasion described extensively in the literature . ProgramAlthough the use of immune checkpoint inhibitors (ICIs), especially those targeting PD-1 and CTLA-4, have demonstrated remarkable efficacy in solid tumors, their application for AML remains an active area of exploration. Several studies attempted to evaluate tumor microenvironment in AML to utilize the ICI in the management of AML . StudiesIn a different study, 23 samples from patients with AML were compared with those of 30 healthy controls. PD-1 expression on CD8 + and CD4+ T cells did not differ significantly compared with healthy controls. Instead, PD-1 was upregulated in peripheral blood samples of patients with AML who relapsed after either intensive chemotherapy or allogeneic stem cell transplantation  compared with those of the same patients at the time of diagnosis. Out of the two relapsed group, the upregulation of PD-1 expression on CD4 and CD8 T cells was more pronounced on its with Allo-SCT . AnotherRanvandi et al.  also invWatts et al.  investigSubklewe et al.  investigUy et al.  investigBispecific T-Cell Engagers (BiTEs) are bispecific antibody constructs that allow for the simultaneous binding of the CD3 receptor on T cells, and tumor-associated antigens on malignant cells. Combining the two different specificities should, in theory, activate exhausted T cells in the TME through sustained tumor antigen exposure. This highly specific process reduces off-target cytotoxicity, as the T cell will only be activated in the presence of malignant cells. Significantly, despite the downregulation of cell-surface antigens by malignant cells to evade the immune system via the downregulation of major histocompatibility complexes (MHC) and costimulatory signals, BiTEs act in an MHC-1 and signal independent manner. This is performed through the formation of a cytolytic synapse between CD8+ T cells regardless of the MHC-1 expression on tumor cells. One issue with the utilization of BiTEs Is the rapid clearance by the kidney, therefore requiring daily continuous infusion to administer the medication. Half-life-extending (HLE) BiTEs, such as AMG673, which include the fusion of an Fc domain, that leads to extension of the half-life, allowing weekly dosing of the agent . The cavDual affinity retargeting antibodies (DARTs) have a disulfide bridge that stabilizes the structure, and results have been encouraging in AML patients, noting an objective response rate ranging between 18% and 30% . AdditioBispecific killer cell engagers (BiKEs) and trispecific killer cell engagers (TriKEs) utilize NK cells via the CD16 receptor, which when activated produces cytokines, such as IL2, that invokes a cytolytic response against the target tumor cells . DespiteSome limitations of BiTE therapy include rapid renal clearance, antigen escape, and nonselective activation of the immune system. Various new constructs have been developed to overcome the rapid clearance, however they come with their own issues. Longer bioavailability leads to increased potential for adverse effects . The mosSuccessful response to immunotherapy is predicated on the immunological composition of the TME. Tumor infiltrating lymphocytes (TILs) represent a heterogeneous population of effector T cells, B cells, and innate lymphoid cells recruited to the TME in response to tumor immune stimulus ,76. AccuThe use of engineered TILs that are expanded ex vivo and reinfused back into pre-conditioned patients has demonstrated remarkable efficacy in treating multiple solid tumors, including melanoma, colorectal, breast, and cervical cancer ,83,84,85The clinical application of TIL therapy in AML may offer several advantages over other immunotherapies being investigated . When co+CD95− or CD62L+ CD45RA+, which are markers for naïve T cells [Using human bone marrow aspirates from AML patients, we recently demonstrated that CD3+ TILs could be isolated and expanded to clinically applicable scales using a novel ex vivo culture system . Expande T cells . As proo T cells . A study T cells . TogetheThe above studies highlight several challenges that will need to be addressed before clinical translation for use in AML. Firstly, we will need to address the reason for variability of TIL populations amongst AML patients. We observed two groups of patients that either displayed low (2.3%) or high numbers (32.6%) of CD3+ TILs, which was consistent with a recent report that CD3 TILS were preserved in 50% of patient samples ,95. FutuChimeric antigen receptor (CAR) therapy involves engineering synthetic receptors to redirect lymphocytes, most frequently T cells, to identify and eliminate cancer cells. The extracellular domain of CARs is typically derived from the single chain variable fragment of monoclonal antibodies which bind to cancer cell antigens. Binding of CAR to the cancer cell antigen initiates intracellular domain signaling through CD3ζ which is suspected to facilitate T cell activation. Simultaneously, a costimulatory domain signaling through 4-1BB or CD28 is suspected to occur which allows T cells to have sustained anti-tumor activity .The highly immunosuppressive AML microenvironment is a challenge to CAR T cell therapy. There are proposed direct and indirect mechanisms contributing to AML immune escape. AML blasts can express the enzymes arginase II and indoleamine 2,3 dioxygenase (IDO) which produce metabolites that hinder T cells while concurrently creating an environment that favors T regulatory cell and MDSC expansion which further dampen CAR T cell response and decrease proliferation. AML blasts may also express ectonucleotidases, such as CD38, CD39, and CD73, that are involved in breaking down ATP and NAD+ to adenosine; the resulting adenosine accumulation leads to T cell suppression. Notably, AML blasts have been reported to directly suppress T cell anti-tumor response by inducing reactive oxygen species (ROS) triggering T cell apoptosis .Despite these challenges, success has been demonstrated in the use of CART cell therapy in AML patients. The first reported clinical trial that demonstrated CAR T cell efficacy in AML was in 2013 . RitchieThe most significant barrier limiting the application of CAR T cell therapy in AML is the absence of an AML-specific antigen. CD33 and CD123 are highly expressed by AML blasts and pre-clinical models have demonstrated CD33 and CD123 directed CAR T cells have highly potent anti-tumor activity ,103. HowThe ultimate application of CAR T therapy in AML is hypothesized in the identification of an AML specific neoantigen that can facilitate cancer cell eradication while sparing normal cells . NeoantiMore evidence indicated that high concentrated intravenous vitamin C showed potential therapeutic effect in cell lines from cancer patients, including patients with AML. There is a dose- and time-dependent inhibition of proliferation in acute myeloid leukemia (AML) cell lines . Phase IThrough the nuclear vitamin D receptor (VDR), 1, 25(OH) 2D3 can attach to the promoter regions of target genes. Many pathways, including MAPKs, JAK/STAT, and PI3K/Akt, are related to 1, 25(OH) 2D3 ,119,120.Compared to the board research of vitamin C and vitamin D, rare articles mentioned about vitamin B and vitamin E and those articles are in vitro studies. Tocopherols and tocotrienols are vitamin E derivatives. In Ghanem’s study, γ-tocotrienol exhibits time and dose-dependent anti-proliferative, pro-apoptotic and antioxidant effects on U937 and KG-1 cell lines (AML cell lines) . AdditioExcept for vitamins, there are lots of natural products that are proven to be related to AML and have the potential to contribute to the idea against the multi-drug resistance of AML cells. Those natural products have different anti-tumor effects, including increasing apoptosis of AML cells, inducing cell cycle arrest, anti-proliferative effects, and even reverse of drug resistance ,143,144.Over the past two decades, the conception of hematologic malignancies as a cell-intrinsic disorder driven by genetic and epigenetic alterations has been revised to include extrinsic mechanisms of resistance.AML is no longer merely viewed as a genetic disease, but rather a complex interplay between leukemic cells with the surrounding cellular and humoral environment. The tumor microenvironment is an important aspect of AML biology, as it contributes to a multitude of mechanisms driving tumorigenesis and resistance to current therapies.Immune-based therapies in AML remain of large interest, despite limited success in clinical trials. The complex immunosuppressive TME likely limits the efficacy of immune-based therapeutic strategies. Thus, it will be critical to consider the AML immune contexture so that we may develop therapies that appropriately redirect the immune microenvironment to favor the elimination of leukemic blasts. Intrinsic and extrinsic factors of AML cells, including genetic mutations and cytokine secretion, affect AML response to immunotherapies. Based on the review of the above literature, it is likely that combination therapy with ICIs, BITE, adoptive T cell therapy (CAR-T or TIL), and natural products will provide more opportunities to achieve sustained clinical remission. Early detection of resistance mechanisms and utilization of strategies to overcome resistance will be the future direction for the development of immunotherapy against AML.


Shwachman-Diamond syndrome (SDS) is a rare bone marrow failure syndrome characterized by exocrine pancreatic insufficiency, bone abnormalities, progressive cytopenia, and predispositions to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). AML, in these patients, is associated with a poor prognosis and with an increased risk of organ toxicity and infectious complications from chemotherapy and hematopoietic stem cell transplantation (HSCT), thus leading to high rates of treatment-related morbidity and mortality. The BCL-2 inhibitor venetoclax has revolutionized the treatment of AML in elderly adults, especially for treatment-naive elderly patients who are ineligible for intensive chemotherapy. There is limited evidence on the use of venetoclax in pediatric patients with SDS-related MDS or AML. Here, we report a case of a 14-year-old boy with SDS with AML arising from MDS. The patient was treated with two cycles of conventional chemotherapy with fludarabine and cytarabine with an initial good response but immediate relapse and substantial toxicity. Treatment with venetoclax and azacitidine was started, with a substantial reduction of leukemic burden . However, it was followed by multiple infectious complications and worsening of the general condition not allowing treatment to be continued, and the patient eventually died from multiorgan failure. With the limitations of observation of a single patient, our experience suggests that venetoclax/azacitidine combination therapy may represent a therapeutic possibility for patients with SDS and AML, even though it may be associated with significant toxicity. Pediatric myelodysplastic syndromes (MDS) are clonal affections of hematopoiesis leading to refractory cytopenia, abnormal maturation of hematopoietic precursors, and risk of evolution to acute myeloid leukemia (AML) . Shwachm2/day and cytarabine 2,000mg/m2/day for 5 days), aiming to achieve leukemic burden reduction in view of HSCT. Treatment was well tolerated overall with no major organ toxicity, yet it was followed by an Escherichia coli sepsis requiring hospital admission , followed by two episodes of sepsis by Staphylococcus epidermidis and Escherichia coli, respectively. An increase of hepatosplenomegaly was also observed, likely due to AML progression, together with marked fluid overload only partially responding to diuretic therapy, and he was considered not eligible for HSCT or to continue therapy with a combination of azacitidine and venetoclax. Palliative therapy with azacitidine alone was started, with no response, and he eventually died from multiorgan failure.We report the case of a 14-year-old boy affected by SDS that had been diagnosed in the first year of life with neutropenia, failure to thrive, and recurrent respiratory infections. In the following years, he had always been well, with moderate-to-severe neutropenia (400–600/mmc) but without severe infections. Yearly hematologic controls were performed, including regular bone marrow aspirates, with no evidence of myelodysplastic progression. When the patient was 16 years and 7 months old, he was admitted for persistent fever without other symptoms. Blood tests showed trilinear cytopenia: therefore, a bone marrow aspirate was performed, which showed a hypercellular marrow with the prevalence of erythroid precursors (50%), often with dysplastic signs, suggesting MDS. A bone marrow biopsy confirmed the presence of a hypercellular MDS with the prevalence of an immature erythroid population and an increase of megakaryocytes, which appeared dysplastic; flow cytometry analysis showed 8% of myeloid blasts (MDS with excess blasts—MDS EB1 according to WHO 2017). The recommendation to proceed to HSCT was formalized, and since he did not have compatible HLA siblings, a search for a matched unrelated donor (MUD) was started without starting any MDS-directed therapy. One month after the diagnosis, while waiting for the HSCT, he developed splenomegaly  and peripheral monocytosis. Repeated bone marrow aspirate showed progression to AML (bone marrow flow cytometry showed 75% blasts CD34+/CD7+/CD45+/CD33+/CD11b dim/CD117+). Cytogenetic and molecular analysis  showed no abnormalities. Treatment with Fludarabine—Cytarabine (FLA) was started (fludarabine 30 mg/m2) with good response and reduction of bone marrow blast percentage to less than 1%. Notably, she underwent cord blood HSCT with primary graft failure and then proceeded to haploidentical HSCT with minimal toxicity after a conditioning regimen consisting of fludarabine, total body irradiation, anti-thymocyte globulin, and cyclophosphamide. In our patient, after the first 2 weeks of combination therapy, we witnessed a complete response in peripheral blast count and a marked reduction of hepatosplenomegaly. Bone marrow re-evaluation after the first month of therapy showed a partial response in relative terms (reduction of blasts from 92% to 52%) but with a marked reduction of AML burden, as the marrow appeared frankly hypocellular. Furthermore, although the response was not complete, the boy had received only one cycle of therapy: experience in adult patients treated with venetoclax/azacitidine for untreated AML showed a complete response after only one cycle in 40% of patients, reaching 66% after the second cycle with a median number of completed 28 days cycles of 7  could possibly have represented the best chance for this patient, albeit with all the limits and caveats associated with the need of using a haploidentical donor. Although haploidentical HSCT has been considered inferior to MUD for a long time, recent advances have led to similar outcomes, making this approach feasible for a wide type of diseases including AML patients . Specifitoxicity . AnotherPatients with SDS that develop MDS and AML are prone to high morbidity and mortality due to severe treatment-related toxicity and relapsed/refractory disease. With the limitations of observation of a single patient, our experience suggests that AML in SDS may respond to venetoclax/azacitidine combination therapy, which could therefore represent an option as a bridge to HSCT. Further studies are necessary to define the best treatment regimens and hopefully shared treatment recommendations for MDS/AML in these patients.