Effects of Age, Colony, and Sex on Mercury Concentrations in California Sea Lions.

We measured total mercury (THg) concentrations in California sea lions (Zalophus californianus) and examined how concentrations varied with age class, colony, and sex. Because Hg exposure is primarily via diet, we used nitrogen (δ (15)N) and carbon (δ (13)C) stable isotopes to determine if intraspecific differences in THg concentrations could be explained by feeding ecology. Blood and hair were collected from 21 adult females and 57 juveniles from three colonies in central and southern California (San Nicolas, San Miguel, and Año Nuevo Islands). Total Hg concentrations ranged from 0.01 to 0.31 µg g(-1) wet weight (ww) in blood and 0.74 to 21.00 µg g(-1) dry weight (dw) in hair. Adult females had greater mean THg concentrations than juveniles in blood (0.15 vs. 0.03 µg(-1) ww) and hair (10.10 vs. 3.25 µg(-1) dw). Age class differences in THg concentrations did not appear to be driven by trophic level or habitat type because there were no differences in δ (15)N or δ (13)C values between adults and juveniles. Total Hg concentrations in adult females were 54 % (blood) and 24 % (hair) greater in females from San Miguel than females from San Nicolas Island, which may have been because sea lions from the two islands foraged in different areas. For juveniles, we detected some differences in THg concentrations with colony and sex, although these were likely due to sampling effects and not ecological differences. Overall, THg concentrations in California sea lions were within the range documented for other marine mammals and were generally below toxicity benchmarks for fish-eating wildlife.

A rapid host-protein test for differentiating bacterial from viral infection: Apollo diagnostic accuracy study.

Objectives: To determine the diagnostic accuracy of a rapid host-protein test for differentiating bacterial from viral infections in patients who presented to the emergency department (ED) or urgent care center (UCC). Methods: This was a prospective multicenter, blinded study. MeMed BV (MMBV), a test based on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), interferon gamma-inducible protein-10 (IP-10), and C-reactive protein (CRP), was measured using a rapid measurement platform. Patients were enrolled from 9 EDs and 3 UCCs in the United States and Israel. Patients >3 months of age presenting with fever and clinical suspicion of acute infection were considered eligible. MMBV results were not provided to the treating clinician. MMBV results (bacterial/viral/equivocal) were compared against a reference standard method for classification of infection etiology determined by expert panel adjudication. Experts were blinded to MMBV results. They were provided with comprehensive patient data, including laboratory, microbiological, radiological and follow-up. Results: Of 563 adults and children enrolled, 476 comprised the study population (314 adults, 162 children). The predominant clinical syndrome was respiratory tract infection (60.5% upper, 11.3% lower). MMBV demonstrated sensitivity of 90.0% (95% confidence interval [CI]: 80.3-99.7), specificity of 92.8% (90.0%-95.5%), and negative predictive value of 98.8% (96.8%-99.6%) for bacterial infections. Only 7.2% of cases yielded equivocal MMBV scores. Area under the curve for MMBV was 0.95 (0.90-0.99). Conclusions: MMBV had a high sensitivity and specificity relative to reference standard for differentiating bacterial from viral infections. Future implementation of MMBV for patients with suspected acute infections could potentially aid with appropriate antibiotic decision-making.

Evaluation of the Panbio™ COVID-19 IgG rapid test device performance.

Background: The Panbio™ COVID-19 IgG Rapid Test Device ("Panbio™") detects IgG antibodies against the SARS-CoV-2 spike protein from viral infection or vaccination. Objectives: To determine the diagnostic sensitivity and specificity of the Panbio™ professional use test, using fingerstick whole blood and venous plasma. Study design: Fingerstick whole blood and venous plasma from each participant were tested with Panbio™ and compared against the SARS-CoV-2 IgG II assay on the Abbott Architect™ platform (Europe) or the equivalent AdviseDx SARS-CoV-2 IgG II Abbott Alinity i™ platform (US). 447 evaluable participants were enrolled across 6 US and 9 European clinical centers. Results: For unvaccinated participants with PCR-confirmed infection ≥21 days post-symptom onset, the Panbio™ sensitivity with fingerstick whole blood was 92.6 % (95 % CI: 85.9, 96.7), and the specificity was 97.0 % (95 % CI: 93.1, 99.0). For venous plasma, the sensitivity was 90.0 % (95 % CI: 79.5, 96.2) for participants with PCR-confirmed infection and symptom onset 22-180 days ago; the specificity was 96.3 % (92.2, 98.6). For vaccinated participants, the sensitivity was 98.4 % (95 % CI: 91.2, 100.0) for fingerstick whole blood and 96.7 % (95 % CI: 88.7, 99.6) for venous plasma. Conclusion: The Panbio™ test had high sensitivity and specificity for detecting IgG against the SARS-CoV-2 spike protein.

The energetic consequences of behavioral variation in a marine carnivore.

Intraspecific variability in foraging behavior has been documented across a range of taxonomic groups, yet the energetic consequences of this variation are not well understood for many species. Understanding the effect of behavioral variation on energy expenditure and acquisition is particularly crucial for mammalian carnivores because they have high energy requirements that place considerable pressure on prey populations. To determine the influence of behavior on energy expenditure and balance, we combined simultaneous measurements of at-sea field metabolic rate (FMR) and foraging behavior in a marine carnivore that exhibits intraspecific behavioral variation, the California sea lion (Zalophus californianus). Sea lions exhibited variability in at-sea FMR, with some individuals expending energy at a maximum of twice the rate of others. This variation was in part attributable to differences in diving behavior that may have been reflective of diet; however, this was only true for sea lions using a foraging strategy consisting of epipelagic (<200 m within the water column) and benthic dives. In contrast, sea lions that used a deep-diving foraging strategy all had similar values of at-sea FMR that were unrelated to diving behavior. Energy intake did not differ between foraging strategies and was unrelated to energy expenditure. Our findings suggest that energy expenditure in California sea lions may be influenced by interactions between diet and oxygen conservation strategies. There were no apparent energetic trade-offs between foraging strategies, although there was preliminary evidence that foraging strategies may differ in their variability in energy balance. The energetic consequences of behavioral variation may influence the reproductive success of female sea lions and result in differential impacts of individuals on prey populations. These findings highlight the importance of quantifying the relationships between energy expenditure and foraging behavior in other carnivores for studies addressing fundamental and applied physiological and ecological questions.

Towards a more precise assay of sperm function in egg binding.

Historically, the treatment of severe male factor infertility has relied on donor sperm insemination. A decade ago the option of treating severe male factor infertility with partner sperm became a viable alternative. With the introduction of intracytoplasmic sperm injection (ICSI) in conjunction with in vitro fertilization (IVF), only men who produce no sperm are denied the option of fathering their own children. The use of ICSI has been extended to couples with mild male factors. Despite the known genetic risks (both inherent and de novo) of ICSI to offspring, couples with male factors as part of their infertility problem often prefer ICSI to standard IVF, due to apprehension that their sperm might not otherwise succeed in fertilization. This apprehension would be alleviated if an assay for the egg binding capability of human sperm were available. We examine here the possibility that recombinant human zona pellucida 3 (rec hZP3), the primary sperm receptor sulfoglycoprotein of the egg zona pellucida (ZP), be used as a human ZP surrogate for assessing sperm ability to bind to the ZP. Unlike human eggs, which cannot be obtained for this purpose, rec hZP3 can be produced in quantity. An efficient assay can be established by incubating sperm with rec hZP3 coated to a microwell plate. Infertile men with sperm having ability to bind to rec hZP3 can be advised to select standard IVF or intrauterine insemination, which have fewer genetic and medical risks.

A 19-year-old man presenting with a generalized body rash.

A 19-year-old man presented to the emergency department with a chief complaint of generalized body rash for two weeks. The rash began shortly after he initiated penicillin therapy for a sore throat diagnosed one week previously. He also complained of having dark urine and abdominal discomfort. His urinalysis revealed proteinuria and hematuria, and he was admitted for further evaluation and management. While in the hospital, he had an episode of hemoptysis. A renal biopsy was performed and revealed IgA deposition. In light of his systemic symptoms including rash and abdominal pain, he was diagnosed with Henoch-Schonlein purpura (HSP).

Acrosin antibodies and infertility. I. Detection of antibodies towards proacrosin/acrosin in women consulting for infertility and evaluation of their effects upon the sperm protease activities.

OBJECTIVE: To detect the presence of antibodies to the proacrosin/acrosin system and to evaluate their effect on the sperm acrosomal protein activities in women consulting for infertility. DESIGN: Retrospective study. SETTING: Basic research laboratory. PATIENT(S): Recombinant proteins derived from human proacrosin (Rec-40, Rec-30, Rec-20, Rec-10) and recombinant human zona pellucida (ZP) glycoprotein A( *) (rec-hZPA). INTERVENTION(S): Development of an ELISA-Acro to test for antiacrosin antibodies using Rec-40 and truncated acrosin proteins as antigens. MAIN OUTCOME MEASURE(S): Evaluation of: 1) the presence of antiacrosin antibodies; 2) the protein regions recognized by the antibodies; 3) the relationship between antiacrosin antibodies and surface antisperm antibodies (ASA) identified by the immunobead binding test (IBT); and 4) the effect of antiacrosin antibodies upon proacrosin/acrosin binding activity to ZPA and acrosin amidase activity. RESULT(S): Antiacrosin antibodies were detected in sera from 34 of 179 women (19%). Detection of ASA by the IBT resulted in a similar incidence (36 of 179, 20%), although only six of them showed correspondence between both assays; five of these six sera were IBT-positive IgGs to the sperm head. Antiacrosin antibodies directed toward different protein regions inhibited proacrosin binding activity to rec-hZPA as well as its activation and acrosin amidase activity in protein sperm extracts. CONCLUSION(S): Antiacrosin antibodies are present in sera of women consulting for infertility in both IBT-positive and IBT-negative samples, and they affect proacrosin/acrosin activities.

Antiacrosin antibodies and infertility. II. Gene immunization with human proacrosin to assess the effect of immunity toward proacrosin/acrosin upon protein activities and animal fertility.

OBJECTIVE: To assess the effect of antiacrosin antibodies upon proacrosin/acrosin activities and animal fertility. DESIGN: Prospective study. SETTING: Basic research laboratory. PATIENT(S): A gene immunization (GI) model was developed; mice were injected with the sequence encoding human proacrosin (h-proacrosin), cloned in an expression vector. INTERVENTION(S): Subcloning of h-proacrosin in a eukaryotic expression vector (promoter, CMV; leader sequence, alpha-1 antitrypsin; pSF2-Acro); GI of female mice with this plasmid. MAIN OUTCOME MEASURE(S): The following parameters were evaluated: [1] adequate conditions for GI protocols, [2] humoral response to GI with pSF2-Acro, [3] protein regions recognized by the antibodies, and [4] effect of antibodies upon proacrosin/acrosin-ZPA binding and amidase activity, and animal fertility. RESULT(S): Conditions of female mice GI with the proacrosin sequence were established (plasmid purification with anion exchange chromatography and 40 microg of pSF2-Acro per dose) to trigger an immune response, reaching maximum levels at week 9 after the first injection. Antibodies produced by GI recognized human and mouse sperm acrosin systems, inhibited human proacrosin/acrosin interaction with recombinant human ZPA and protease activity, and negatively affected mouse IVF and early embryonic development. In addition, mice immunized with SF2-Acro exhibited a significantly lower size of fetuses. CONCLUSION(S): Antiacrosin antibodies developed by using GI inhibit human proacrosin/acrosin activities and impair mouse fertility.

Recombinant human zona pellucida protein C produced in Chinese hamster ovary cells binds to human spermatozoa and inhibits sperm-zona pellucida interaction.

Recombinant human zona pellucida protein C expressed in Chinese hamster ovary cells associates to the acrosomal region of human spermatozoa and inhibits sperm-zona pellucida interaction in the hemizona assay. Recombinant human zona pellucida protein C may be a useful tool toward the development of diagnostic methods for male factor infertility and the elucidation of the molecular basis of fertilization.

Binding of recombinant human proacrosin/acrosin to zona pellucida (ZP) glycoproteins. I. Studies with recombinant human ZPA, ZPB, and ZPC.

OBJECTIVE: To characterize proacrosin/acrosin interaction with isolated zona pellucida (ZP) components. DESIGN: Prospective study. SETTING: Basic research laboratory. PATIENT(S): Recombinant proteins derived from human proacrosin (Rec-40, Rec-30, Rec-20, Rec-10, and Rec-6) and from human ZP glycoproteins (rec-hZPA, ZPB, and ZPC). INTERVENTION(S): In vitro binding assay developed to assess proacrosin/acrosin-ZP interaction. MAIN OUTCOME MEASURE(S): Zona pellucida glycoprotein binding to proacrosin/acrosin; estimation of binding affinity. RESULT(S): Of all ZP proteins, rec-hZPA demonstrated the highest binding activity toward acrosin (Rec-30) (rec-hZPB: 42% of rec-hZPA; rec-hZPC: 39% of rec-hZPA; P<.0005). Rec-hZPA interaction was disturbed by dextran sulphate (75% inhibition with 10 microM), fucose (67% inhibition with 1.5 microM), and mannose (69% inhibition with 333 mM). Comparing binding activity of proacrosin with other N-terminal acrosin fragments, Rec-40 showed 2.6-3 times higher levels. Moreover, saturable high affinity binding of Rec-40 to ZP components was observed (Kd: 34 nM for rec-hZPA, 38 nM for rec-hZPB, 63 nM for rec-hZPC). CONCLUSION(S): The rec-hZPA is the major ZP ligand for human proacrosin/acrosin. The interaction involves mannosyl, fucosyl, and sulfated glycans. Binding sites for rec-hZP would be located both at the N- and C-terminus of proacrosin, revealing a key role of the proenzyme in the interaction.

Cloning and expression of cynomolgus monkey and baboon zona pellucida proteins.

Partial clones for the three cynomolgus monkey (Macaca fasicularis) zona pellucida genes (cmZPA, cmZPB, and cmZPC) have previously been isolated. These partial clones contained the sequences for the C-terminal portion of each rcmZP protein. To obtain full-length clones for each cmZP, a fresh cynomolgus monkey ovarian cDNA library was constructed. PCR methodology was employed to speed the isolation of full-length clones for each cmZP cDNA. The 3' primers were designed based on sequence information from the previously identified clones; the 5' primers were designed using the human ZP sequences. The PCR technique yielded full-length clones of cmZPA and cmZPC, but not of cmZPB. Therefore, a genomic clone of cmZPB was isolated and the sequence determined. The exon/intron structure is nearly identical to the human ZPB exon/intron structure. New PCR primers were designed based on the cynomolgus monkey ZPB genomic sequence, and a full-length cmZPB cDNA was obtained. The same primers that were used to generate the cmZPB were also used to generate a baboon (Papio cynocephalus) ZPB (bZPB) cDNA. As was done previously for the human zona pellucida (hZP) cDNAs, the cmZP, and bZPB cDNAs were transferred to shuttle vectors for transfection into Chinese Hamster Ovary (CHO) cells. Stable cell lines for producing each ZP protein were isolated. Each cell line secreted the desired recombinant zona pellucida (rZP) protein into the culture medium, and each protein was purified using an established protocol. In terms of size and purity, the purified recombinant cmZP (rcmZP) and rbZPB proteins resemble the rhZP proteins.

A role for the human sperm glycine receptor/Cl(-) channel in the acrosome reaction initiated by recombinant ZP3.

Previously, we have demonstrated an essential role for the neuronal glycine receptor (GlyR) in the acrosome reaction (AR) of mouse and porcine sperm initiated by the egg zona pellucida (ZP). In the present study, we have demonstrated presence of the GlyR in human sperm by immunoprecipitation and Western blot analysis, investigated the potential of a recombinant human ZP3 (rhZP3) preparation as an alternative research tool to solubilized human ZP, and shown that the human sperm GlyR is essential to the human AR initiated by rhZP3. Additionally, we have been able to demonstrate that rhZP3 possesses biological activity, because it is able to rapidly stimulate the AR in capacitated human sperm and its action is blocked by the addition of pertussis toxin. Moreover, spectrofluorometric studies using fura-2-loaded human sperm have shown that rhZP3 triggers a peak-and-plateau rise in intracellular Ca(2+) levels similar to that seen with solubilized mammalian ZP. These results suggest that the actions of rhZP3 and solubilized ZP are elicited via the same signal transduction pathways. Furthermore, incubation of human sperm with an antibody directed against the alpha1 subunit of the human spinal cord GlyR or with 50 nM strychnine caused significant inhibition in the rhZP3-initated AR. Finally, studies using fura-2-loaded human sperm showed that 50 nM strychnine was also able to inhibit the Ca(2+) influx associated with addition of rhZP3. These results further support the view that rhZP3 and the ZP work through the same mechanisms, show that the GlyR is involved in rhZP3-initiated AR, and suggest that the GlyR may also play a role in the early signal transduction cascades associated with ZP-initiated AR in vivo.