Subterranean freshwater gastropod biodiversity and conservation in the United States and Mexico.

Many taxonomic groups successfully exploit groundwater environments and have adapted to a subterranean (stygobiotic) existence. Among these groups are freshwater gastropods (stygosnails), which represent a widespread and taxonomically diverse component of groundwater ecosystems in North America. However, owing to sampling difficulty and lack of targeted study, stygosnails remain among the most understudied of all subterranean groups. We conducted a literature review to assess the biodiversity and geographic associations of stygosnails, along with the threats, management activities, and policy considerations related to the groundwater systems they inhabit. We identified 39 stygosnail species known to occur in a range of groundwater habitats from karst regions in the United States and Mexico. Most stygosnails exhibit extreme narrow-range endemism, resulting in a high risk of extinction from a single catastrophic event. We found that anthropogenically driven changes to surface environments have led to changes in local hydrology and degradation of groundwater systems inhabited by stygosnails such as increased sedimentation, introduction of invasive species, groundwater extraction, or physical collapse of water-bearing passages. Consequently, 32 of the 39 described stygosnail species in the United States and Mexico have been assessed as imperiled under NatureServe criteria, and 10 species have been assessed as threatened under International Union for Conservation of Nature criteria. Compared with surface species of freshwater snails, stygosnail conservation is uniquely hindered by difficulties associated with accessing subterranean habitats for monitoring and management. Furthermore, only three species were found to have federal protection in either the United States or Mexico, and current laws regulating wildlife and water pollution at the state and federal level may be inadequate for protecting stygosnail habitats. As groundwater systems continue to be manipulated and relied on by humans, groundwater-restricted fauna such as stygosnails should be studied so unique biodiversity can be protected.

Diversidad y Conservación de Gasterópodos Subterráneos de Agua Dulce en los Estados Unidos y en México Resumen Muchos grupos taxonómicos aprovechan exitosamente los ambientes de aguas subterráneas y se han adaptado eficazmente a una existencia subterránea (estigobiótica). Entre estos grupos están los gasterópodos (estigocaracoles), los cuales representan un componente taxonómicamente diverso y de amplia distribución en los ecosistemas de aguas subterráneas en América del Norte. Sin embargo, debido a la dificultad del muestreo y a la falta de estudios enfocados, los estigocaracoles todavía son de los grupos menos estudiados de los taxones subterráneos. Realizamos una revisión de la literatura para evaluar las asociaciones geográficas y la biodiversidad de los estigocaracoles, junto con las amenazas, actividades de manejo y consideraciones políticas relacionadas con los sistemas de aguas subterráneas que habitan. Identificamos a 39 especies de estigocaracoles que se sabe se encuentran en una gama de hábitats de aguas subterráneas de las regiones kársticas en los Estados Unidos y en México. La mayoría de los estigocaracoles exhiben un endemismo extremo de extensión limitada, lo que resulta en un riesgo elevado de extinción a partir de un evento catastrófico único. Descubrimos que los cambios causados por el hombre en los ambientes superficiales han resultado en cambios en la hidrología local y en la degradación de los sistemas de aguas subterráneas habitadas por los estigocaracoles. Dichos cambios incluyen incremento de la sedimentación, la introducción de especies invasoras, la extracción de aguas subterráneas y el colapso físico de los pasos de agua. Como consecuencia, 32 de las 39 especies descritas de estigocaracoles en los Estados Unidos y en México han sido valoradas como en peligro bajo los criterios de NatureServe, y diez especies han sido valoradas como amenazadas bajo los criterios de la Unión Internacional para la Conservación de la Naturaleza. Comparada con las especies superficiales de caracoles de agua dulce, la conservación de los estigocaracoles está singularmente obstaculizada por las dificultades asociadas con el acceso a los hábitats subterráneos para su monitoreo y manejo. Además, se encontró que sólo tres especies cuentan con protección federal ya sea en Estados Unidos o en México, y puede que las leyes actuales que regulan la vida silvestre y la contaminación del agua a nivel estatal y federal sean inadecuadas para la protección de los hábitats de los estigocaracoles. Mientras los sistemas de aguas subterráneas sigan siendo manipulados y los humanos sigan dependiendo de ellos, la fauna restringida a las aguas subterráneas, como los estigocaracoles, debería ser estudiada para proteger a la biodiversidad tan única.

Chemolithoautotrophy supports macroinvertebrate food webs and affects diversity and stability in groundwater communities.

The prevailing paradigm in subterranean ecology is that below-ground food webs are simple, limited to one or two trophic levels, and composed of generalist species because of spatio-temporally patchy food resources and pervasive energy limitation. This paradigm is based on relatively few studies of easily accessible, air-filled caves. However, in some subterranean ecosystems, chemolithoautotrophy can subsidize or replace surface-based allochthonous inputs of photosynthetically derived organic matter (OM) as a basal food resource and promote niche specialization and evolution of higher trophic levels. Consequently, the current subterranean trophic paradigm fails to account for variation in resources, trophic specialization, and food chain length in some subterranean ecosystems. We reevaluated the subterranean food web paradigm by examining spatial variation in the isotopic composition of basal food resources and consumers, food web structure, stygobiont species diversity, and chromophoric organic matter (CDOM), across a geochemical gradient in a large and complex groundwater system, the Edwards Aquifer in Central Texas (USA). Mean δ13C values of stygobiont communities become increasingly more negative along the gradient of photosynthetic OM sources near the aquifer recharge zone to chemolithoautotrophic OM sources closer to the freshwater-saline water interface (FWSWI) between oxygenated freshwater and anoxic, sulfide-rich saline water. Stygobiont community species richness declined with increasing distance from the FWSWI. Bayesian mixing models were used to estimate the relative importance of photosynthetic OM and chemolithoautorophic OM for stygobiont communities at three biogeochemically distinct sites. The contribution of chemolithoautotrophic OM to consumers at these sites ranged between 25% and 69% of total OM utilized and comprised as much as 88% of the diet for one species. In addition, the food web adjacent to the FWSWI had greater trophic diversity when compared to the other two sites. Our results suggest that diverse OM sources and in situ, chemolithoautotrophic OM production can support complex groundwater food webs and increase species richness. Chemolithoautotrophy has been fundamental for the long-term maintenance of species diversity, trophic complexity, and community stability in this subterranean ecosystem, especially during periods of decreased photosynthetic production and groundwater recharge that have occurred over geologic time scales.

Synthesis of site-specific antibody-drug conjugates using unnatural amino acids.

Antibody-drug conjugates (ADCs) allow selective targeting of cytotoxic drugs to cancer cells presenting tumor-associated surface markers, thereby minimizing systemic toxicity. Traditionally, the drug is conjugated nonselectively to cysteine or lysine residues in the antibody. However, these strategies often lead to heterogeneous products, which make optimization of the biological, physical, and pharmacological properties of an ADC challenging. Here we demonstrate the use of genetically encoded unnatural amino acids with orthogonal chemical reactivity to synthesize homogeneous ADCs with precise control of conjugation site and stoichiometry. p-Acetylphenylalanine was site-specifically incorporated into an anti-Her2 antibody Fab fragment and full-length IgG in Escherichia coli and mammalian cells, respectively. The mutant protein was selectively and efficiently conjugated to an auristatin derivative through a stable oxime linkage. The resulting conjugates demonstrated excellent pharmacokinetics, potent in vitro cytotoxic activity against Her2(+) cancer cells, and complete tumor regression in rodent xenograft treatment models. The synthesis and characterization of homogeneous ADCs with medicinal chemistry-like control over macromolecular structure should facilitate the optimization of ADCs for a host of therapeutic uses.

Self-assembled antibody multimers through peptide nucleic acid conjugation.

With the recent clinical success of bispecific antibodies, a strategy to rapidly synthesize and evaluate bispecific or higher order multispecific molecules could facilitate the discovery of new therapeutic agents. Here, we show that unnatural amino acids (UAAs) with orthogonal chemical reactivity can be used to generate site-specific antibody-oligonucleotide conjugates. These constructs can then be self-assembled into multimeric complexes with defined composition, valency, and geometry. With this approach, we generated potent bispecific antibodies that recruit cytotoxic T lymphocytes to Her2 and CD20 positive cancer cells, as well as multimeric antibody fragments with enhanced activity. This strategy should accelerate the synthesis and in vitro characterization of antibody constructs with unique specificities and molecular architectures.

Synthesis of bispecific antibodies using genetically encoded unnatural amino acids.

Bispecific antibodies were constructed using genetically encoded unnatural amino acids with orthogonal chemical reactivity. A two-step process afforded homogeneous products in excellent yield. Using this approach, we synthesized an anti-HER2/anti-CD3 bispecific antibody, which efficiently cross-linked HER2+ cells and CD3+ cells. In vitro effector-cell mediated cytotoxicity was observed at picomolar concentrations.

New species of Pyrgulopsis Call & Pilsbry, 1886 (Mollusca: Caenogastropoda: Hydrobiidae) from two Chihuahuan Desert springs.

Two new populations of Pyrgulopsis were found in springs in the Chihuahuan desert region of Texas. Pyrgulopsis Call & Pilsbry, 1886 are a diverse group of snails occupying freshwater springs, usually narrow-range endemics limited to a single spring. Their small ranges confer substantial conservation concern as the drying or disturbance of a single spring can lead to their extinction. We use mitochondrial COI and nuclear LSU sequences, shell morphometrics, and anatomical features to distinguish a new species of Pyrgulopsis endemic to the Big Bend region of Texas.

Hyporheic ostracods (Crustacea, Ostracoda) from Texas (USA) with six new species.

Between 2015 and 2017, 58 ostracod taxa were collected at 30 locations in 111 samples from the hyporheic zone of surface streams in Texas, U.S.A. Six new species (Bradleycypris foresteri n. sp., Bradleystrandesia macula n. sp., Pseudocandona lordi n. sp., Indocandona rusti n. sp., Stenocypris sancari n. sp., Cyclocypris dalyana n. sp.) are described based on carapace and soft body parts. Additionally, 17 species and two genera (Vestalenula and Indocandona) are presented as new records for Texas, and Indocandona rusti n. sp. is the first record of the genus outside India. Taxonomic positions of the new species are discussed along with ecological information. One-hundred and six nonmarine ostracod species are now reported from Texas. However, ongoing studies suggest that actual species richness is much higher.

Two new species of Pyrgulopsis Call amp; Pilsbry, 1886 (Mollusca: Caenogastropoda: Hydrobiidae) from springs in the Rio Grande watershed in Texas.

Pyrgulopsis Call Pilsbry, 1886 is a genus of small (<5 mm) spring snails, usually endemic to single freshwater springs. Two new populations of Pyrgulopsis found in very small, isolated springs and spring runs in the mainstem Rio Grande watershed of western Texas are distinguished from congeners. Mitochondrial and nuclear sequences, morphometrics, and morphological characteristics support Pyrgulopsis rubra sp. nov. and Pyrgulopsis harrymilleri sp. nov. as distinct from other known Pyrgulopsis species, including the geographically proximate P. metcalfi.

Cirolanides wassenichae sp. nov., a freshwater, subterranean Cirolanidae (Isopoda, Cymothoida) with additional records of other species from Texas, United States.

Cirolanides wassenichae sp. nov., is described from the phreatic zone of the Edwards Aquifer, Texas, USA where it is sympatric with Cirolanides texensis Benedict, 1896. Its status as a new species is based on both morphological and molecular data. Number of antennula articles (3-5 vs 9-15), size (mean sizes of 9.5 and 8.8 mm vs 11.1 and 10.4 mm for males and females, respectively), morphology of pereopods 1-3 (haptorial to semi-haptorial in 1-3 vs only 1 haptorial), and shape of pleotelson (squared, slightly indented vs rounded) are key morphological characteristics that distinguish C. wassenichae sp. nov. from C. texensis. Phylogenies based on cytochrome oxidase 1 and large ribosomal subunit 28S show that divergent morphologies correspond to reciprocally monophyletic groups for both nuclear and mitochondrial datasets. The genus Cirolanides is in need of revision, as our description of C. wassenichae sp. nov. renders C. texensis paraphyletic.

Cypria lacrima sp. nov. A New Ostracoda (Candonidae, Crustacea) Species from Texas, U.S.A.

Okan Külköylüoglu, Derya Akdemir, Mehmet Yavuzatmaca, Benjamin F. Schwartz, and Benjamin T. Hutchins (2017) A new ostracod species, Cypria lacrima sp. nov. (Cyclocypridinae) is proposed from an artesian well in Hays County, Texas, U.S.A. The species differs from its conspecies with the following characteristics: unique shape and pit ornamentation on carapace, absence of "e" and "g" setae on both walking and cleaning legs, long gamma seta on Mandibular palp, asymmetric clasping organs and differences in the shape and size of other cheatotaxial parts. A detailed comparison among the species of the genus is provided along with ecological implications. This report increases the geographic distribution of the genus in the southern USA.

Lacrimacandona n. gen. (Crustacea: Ostracoda: Candonidae) from the Edwards Aquifer, Texas (USA).

Lacrimacandona n. gen. with its type species Lacrimacandona wisei n. sp. from the San Marcos artesian well on the Texas State University campus (Hays County, Texas, USA) is proposed as a new genus of the subfamily Candoninae. The new species is sexually dimorphic, and so far no congeneric species have been found. The new genus has the following distinguishing characters: subtriangular carapace tapering posteriorly, uropod with a claw-like anterior seta and two short claws, one long "a" setae on male maxilliped, very large asymmetric clasping organs in males, unique shape and size of hemipenis, and unique occurrence of setae on segments.

SMI-Ribosome inactivating protein conjugates selectively inhibit tumor cell growth.

Cell-targeting conjugates of Saporin 6, a ribosome inactivating protein (RIP), were prepared using the Saporin Ala 157 Cys mutant, a small molecule inhibitor (SMI) of integrins αvß3/αvß5, and a potent cytotoxin, auristatin F (AF). The conjugates selectively and potently inhibited proliferation of tumor cells expressing the target integrins. We anticipate that the small molecule-RIP bioconjugate approach can be broadly applied using other small molecule drugs.

Site-specific coupling and sterically controlled formation of multimeric antibody fab fragments with unnatural amino acids.

Immunoconjugates and multispecific antibodies are rapidly emerging as highly potent experimental therapeutics against cancer. We have developed a method to incorporate an unnatural amino acid, p-acetylphenylalanine (pAcPhe) into an antibody antigen binding fragment (Fab) targeting HER2 (human epidermal growth factor receptor 2), allowing site-specific labeling without disrupting antigen binding. Expression levels of the pAcPhe-containing proteins were comparable to that of wild-type protein in shake-flask and fermentation preparations. The pAcPhe-Fabs were labeled by reaction with hydroxylamine dye and biotin species to produce well-defined, singly conjugated Fabs. We then coupled a hydroxylamine biotin to the pAcPhe-Fab and demonstrated controlled assembly of Fabs in the presence of the tetrameric biotin-binding protein, NeutrAvidin. The position of Fab biotinylation dictates the geometry of multimer assembly, producing unique multimeric Fab structures. These assembled Fab multimers differentially attenuate Her2 phosphorylation in breast cancer cells that overexpress the Her2 receptor. Thus, an encoded unnatural amino acid produces a chemical "handle" by which immunoconjugates and multimers can be engineered.

Selective formation of covalent protein heterodimers with an unnatural amino acid.

We report a strategy for the generation of heterodimeric protein conjugates using an unnatural amino acid with orthogonal reactivity. This paper addresses the challenges of site-specificity and homogeneity with respect to the synthesis of bivalent proteins and antibody-drug conjugates. There are numerous antibody-drug conjugates in preclinical and clinical development, yet these are based either on nonspecific lysine coupling chemistry or on disulfide modification made difficult by the large number of cysteines in antibodies. Here, we describe a recombinant approach that can be used to rapidly generate a variety of constructs with defined conjugation sites. Moreover, this methodology results in homogeneous antibody conjugates whose biological, physical, and pharmacological properties can be quantitatively assessed and subsequently optimized. As proof of concept, we have generated anti-Her2 Fab-Saporin conjugates that demonstrate excellent potency in vitro.

Magnet assisted fabrication of microtubule arrays.

Magnetic fields were used to fashion permanently aligned microtubule arrays allowing formation of highly ordered platforms for assembly, separation, and single molecule motor studies.