Microfluidic-based 18F-labeling of biomolecules for immuno-positron emission tomography

Methods for tagging biomolecules with fluorine 18 as immuno-positron emission tomography (immunoPET) tracers require tedious optimization of radiolabeling conditions and can consume large amounts of scarce biomolecules. We describe an improved method using a digital microfluidic droplet generation (DMDG) chip, which provides computer-controlled metering and mixing of 18F tag, biomolecule, and buffer in defined ratios, allowing rapid scouting of reaction conditions in nanoliter volumes. The identified optimized conditions were then translated to bench-scale 18F labeling of a cancer-specific engineered antibody fragments, enabling microPET imaging of tumors in xenografted mice at 0.5 to 4 hours postinjection.     

Estuarine fouling communities are dominated by nonindigenous species in the presence of an invasive crab

Interactions between anthropogenic disturbances and introduced and native species can shift ecological communities, potentially leading to the successful establishment of additional invaders. Since its discovery in New Jersey in 1988, the Asian shore crab (Hemigrapsus sanguineus) has continued to expand its range, invading estuarine and coastal habitats in eastern North America. In estuarine environments, H. sanguineus occupies similar habitats to native, panopeid mud crabs. These crabs, and a variety of fouling organisms (both NIS and native), often inhabit man-made substrates (like piers and riprap) and anthropogenic debris. In a series of in situ experiments at a closed dock in southwestern Long Island (New York, USA), we documented the impacts of these native and introduced crabs on hard-substrate fouling communities. We found that while the presence of native mud crabs did not significantly influence the succession of fouling communities compared to caged and uncaged controls, the presence of introduced H. sanguineus reduced the biomass of native tunicates (particularly Molgula manhattensis), relative to caged controls. Moreover, the presence of H. sanguineus favored fouling communities dominated by introduced tunicates (especially Botrylloides violaceous and Diplosoma listerianum). Altogether, our results suggest that H. sanguineus could help facilitate introduced fouling tunicates in the region, particularly in locations where additional solid substrates have created novel habitats.     

ACUTE EFFECTS OF A RESISTED DYNAMIC WARM-UP PROTOCOL ON JUMPING PERFORMANCE

This study aimed to investigate the kinematic and kinetic changes when resistance is applied in horizontal and vertical directions, produced by using different percentages of body weight, caused by jumping movements during a dynamic warm-up. The group of subjects consisted of 35 voluntary male athletes (19 basketball and 16 volleyball players; age: 23.4 ± 1.4 years, training experience: 9.6 ± 2.7 years; height: 177.2 ± 5.7 cm, body weight: 69.9 ± 6.9 kg) studying Physical Education, who had a jump training background and who were training for 2 hours, on 4 days in a week. A dynamic warm-up protocol containing seven specific resistance movements with specific resistance corresponding to different percentages of body weight (2%, 4%, 6%, 8%, 10%) was applied randomly on non consecutive days. Effects of different warm-up protocols were assessed by pre-/post- exercise changes in jump height in the countermovement jump (CMJ) and the squat jump (SJ) measured using a force platform and changes in hip and knee joint angles at the end of the eccentric phase measured using a video camera. A significant increase in jump height was observed in the dynamic resistance warm-up conducted with different percentages of body weight (p < 0.05). On the other hand, no significant difference in different percentages of body weight states was observed (p > 0.05). In jump movements before and after the warm-up, while no significant difference between the vertical ground reaction forces applied by athletes was observed (p > 0.05), in some cases of resistance, a significant reduction was observed in hip and knee joint angles (p < 0.05). The dynamic resistance warm-up method was found to cause changes in the kinematics of jumping movements, as well as an increase in jump height values. As a result, dynamic warm-up exercises could be applicable in cases of resistance corresponding to 6-10% of body weight applied in horizontal and vertical directions in order to increase the jump performance acutely.     

Synthesis and binding properties of deltorphin I analogues containing (R) and (S)-alpha-hydroxymethylnaphtylalanine

New analogues of deltorphin I (DT I), in which the phenylalanine residue in position 3 is substituted with amphiphilic alpha,alpha-disubstituted amino acid enantiomers, (R) and (S)-alpha-hydroxymethylnaphtylalanine, were synthesized and tested for mu and delta opioid receptor affinity and selectivity. Although both analogues have lower affinity to delta receptors than DT I, they both expressed specificity to delta receptors.     

Multifactorial diversity sustains microbial community stability

Maintenance of a high degree of biodiversity in homogeneous environments is poorly understood. A complex cheese starter culture with a long history of use was characterized as a model system to study simple microbial communities. Eight distinct genetic lineages were identified, encompassing two species: Lactococcus lactis and Leuconostoc mesenteroides. The genetic lineages were found to be collections of strains with variable plasmid content and phage sensitivities. Kill-the-winner hypothesis explaining the suppression of the fittest strains by density-dependent phage predation was operational at the strain level. This prevents the eradication of entire genetic lineages from the community during propagation regimes (back-slopping), stabilizing the genetic heterogeneity in the starter culture against environmental uncertainty.     

Analogs of arginine vasopressin modified in position 3 with (R)-alpha-hydroxymethylphenylalanine.

This study describes the synthesis and some pharmacological properties of three new analogs of arginine vasopressin (AVP) substituted in position 3 with (R)-alpha-hydroxymethylphenylalanine ([R]-HmPhe). All new peptides were tested for vasopressor and antidiuretic as well as uterotonic activity. None of the 3 analogs showed any pressor activity and their uterotonic activity was negligible. Only analog [Mpa1,(R)-HmPhe3]AVP exhibited significant antidiuretic activity.     

The Cul3–KLHL21 E3 ubiquitin ligase targets Aurora B to midzone microtubules in anaphase and is required for cytokinesis

Cul3 (Cullin3)-based E3 ubiquitin ligases recently emerged as critical regulators of mitosis. In this study, we identify two mammalian BTB (Bric-a-brac–Tramtrack–Broad complex)-Kelch proteins, KLHL21 and KLHL22, that interact with Cul3 and are required for efficient chromosome alignment. Interestingly, KLHL21 but not KLHL22 is necessary for cytokinesis and regulates translocation of the chromosomal passenger complex (CPC) from chromosomes to the spindle midzone in anaphase, similar to the previously described BTB-Kelch proteins KLHL9 and KLHL13. KLHL21 directly binds to Aurora B and mediates ubiquitination of Aurora B in vitro. In contrast to KLHL9 and KLHL13, KLHL21 localizes to midzone microtubules in anaphase and recruits Aurora B and Cul3 to this region. Together, our results suggest that different Cul3 adaptors nonredundantly regulate Aurora B during mitosis, possibly by ubiquitinating different pools of Aurora B at distinct subcellular localizations.     

Identification of bacteria in blood culture broths using matrix-assisted laser desorption-ionization Sepsityper™ and time of flight mass spectrometry

Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) is a novel method for the direct identification of bacteria from blood culture broths. We evaluate for the first time, the performance of the MALDI Sepsityper? Kit and MS for the identification of bacteria compared to standard phenotypic methods using the manufacturer's specified bacterial identification criteria (spectral scores ≥1.700-1.999 and ≥2.000 indicated identification to genus and species level, respectively). Five hundred and seven positive blood culture broths were prospectively examined, of which 379 (74.8%; 358 monomicrobial, 21 polymicrobial) were identified by MALDI-TOF MS; 195 (100%) and 132 (67.7%) of 195 gram-positive; and 163 (100%) and 149 (91.4%) of 163 gram-negative organisms from monomicrobial blood cultures were correctly identified to genus and species level, respectively. Spectral scores <1.700 (no identification) were obtained in 128/507 (25.2%) positive blood culture broths, including 31.6% and 32.3% of gram-positive and polymicrobial blood cultures, respectively. Significantly more gram-negative organisms were identified compared to gram-positive organisms at species level (p<0.0001). Five blood cultures were misidentified, but at species level only; including four monomicrobial blood cultures with Streptococcus oralis/mitis that were misidentified as Streptococcus pneumoniae. Positive predictive values for the direct identification of both gram-positive and gram-negative bacteria from monomicrobial blood culture broths to genus level were 100%. A diagnostic algorithm for positive blood culture broths that incorporates gram staining and MALDI-TOF MS should identify the majority of pathogens, particularly to genus level.     

Position Three in Vasopressin Antagonist Tolerates Conformationally Restricted and Aromatic Amino Acid Substitutions: A Striking Contrast with Vasopressin Agonists

We report the solid-phase synthesis and some pharmacological properties of 12 position three modified analogues (peptides 1–12) of the potent non-selective antagonist of the antidiuretic (V₂-receptor), vasopressor (V_1a-receptor) responses to arginine vasopressin (AVP) and of the uterine contracting (OT-receptor) responses to oxytocin (OT), [1(-β mercapto-β,β-pentamethy lenepropionic acid)-2-O-ethyl-d -tyrosine 4-valine] arginine vasopressin [d(CH₂)₅D -Tyr(Et) ²VAVP] (A) and two analogues of ( B ) (peptides 13,14), the 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid³ (Tic³) analogue of ( A ). Peptides 1–12 have the following substituents at position three in ( A ): (1) Pro; (2) Oic; (3) Atc; (4) D -Atc; (5) Aic; (6) D -Phe; (7) Ile; (8) Leu; (9) Tyr; (10) Trp; (11) Hphe; (12) [HO]Tic; Peptide (13) is the Tyr-NH₂ ⁹ analogue of ( B ): Peptide (14) is the D -Cys ⁶ analogue of ( B ). All 14 new peptides were evaluated for agonistic and antagonistic activities in in vivo V₂ and V_1a assays and in in vitro (no Mg²⁺) _n oxytocic assays. With the exception of the D -Phe³ peptide (No. 6), which exhibits very weak V₂ agonism (…0.0017 u/mg), none of the remaining 13 peptides exhibit any agonistic activities in these assays. In striking contrast to their deleterious effects on agonistic activities in AVP, the Pro³, Oic³, Tyr³, Trp³ and Hphe³ substitutions in ( A ) are very well tolerated, leading to excellent retention of V₂, V_1a and OT antagonistic potencies. All are more potent as V₂ antagonists than the Ile³ and Leu³ analogues of ( A ). The Tyr-NH₂⁹ and D -Cys⁶ substitutions in ( B ) are also well tolerated. The anti-V₂ pA₂ values of peptides 1–5 and 7–14 are as follows (1) 7.77±0.03; (2) 7.41± 0.05; (3) 6.86±0.02; (4) 5.66±0.09; (5) …5.2; (7) 7.25± 0.08; (8) 6.82±0.06; (9) 7.58±0.05; (10) 7.61±0.08; (11) 7.59±0.07; (12) 7.20±0.05; (13) 7.57±0.1; (14) 7.52± 0.06. All analogues antagonize the vasopressor responses to AVP, with anti-V _1a pA₂ values ranging from 5.62 to 7.64, and the in vitro responses to OT, with anti-OT pA₂ values ranging from 5.79 to 7.94. With an anti-V₂ potency of 7.77±0.03, the Pro³ analogue of ( A ) is surprisingly equipotent with ( A ), (anti-V₂ pA₂=7.81±0.07). These findings clearly indicate that position three in AVP V₂/V_1a antagonists, in contrast to position three in AVP agonists, is much more amenable to structural modification than had heretofore been anticipated. Furthermore, the surprising retention of V₂ antagonism exhibited by the Pro³, Oic³, Tyr³, Trp³ and Hphe³ analogues of ( A ), together with the excellent retention of V₂ antagonism by the Tyr-NH₂⁹ and D -Cys⁶ analogues of ( B ) are promising new leads to the design of potent and possibly orally active V₂ antagonists for use as pharmacological tools and/or as radioiodinatable ligands and for development as potential therapeutic agents for the treatment of the hyponatremia caused by the syndrome of the inappropriate secretion of the antidiuretic hormone (SIADH). © 1997 European Peptide Society and John Wiley & Sons, Ltd.