Neuronal GPCR NMUR-1 regulates energy homeostasis in response to pathogen infection.

A key question in current immunology is how the innate immune system generates high levels of specificity. Our previous study in Caenorhabditis elegans revealed that NMUR-1, a neuronal G protein-coupled receptor homologous to mammalian receptors for the neuropeptide neuromedin U (NMU), regulates distinct innate immune responses to different bacterial pathogens. Here, by using quantitative proteomics and functional assays, we discovered that NMUR-1 regulates F 1 F O ATP synthase and ATP production in response to pathogen infection, and that such regulation contributes to NMUR-1-mediated specificity of innate immunity. We further demonstrated that ATP biosynthesis and its contribution to defense is neurally controlled by the NMUR-1 ligand CAPA-1 and its expressing neurons ASG. These findings indicate that NMUR-1 neural signaling regulates the specificity of innate immunity by controlling energy homeostasis as part of defense against pathogens. Our study provides mechanistic insights into the emerging roles of NMU signaling in immunity across animal phyla.

Language Accommodations for Limited English Proficient Patients in Rural Health Care.

Over 25 million individuals living in America are limited English proficient, many of whom live in rural communities. Adequate language accommodations are critical to providing effective healthcare for these populations. An online questionnaire was delivered to 42 rural facilities in Washington State. It included questions about their demand for language services, modalities of interpretation, translated documentation and barriers to providing accommodations. Fifteen of 42 (35.7%) responded. Spanish, Russian, Vietnamese, Japanese, Ukrainian and Mam were encountered daily. Telephonic and virtual remote interpreter services were the most widely available. Not all facilities had vital documents translated to frequently encountered languages. Challenges to providing language access were reported by nearly all participants. The rural facilities surveyed all encountered LEP patient populations and offered oral interpretation. Overall, these facilities were meeting requirements for providing language accommodation services. Even so, many facilities reported experiencing barriers to providing these services.

Hospital-level variation in the quality of urologic cancer surgery.

BACKGROUND: Unexplained variation in outcomes after common surgeries raises concerns about the quality and appropriateness of surgical care. Understanding variation in surgical outcomes may identify processes that could affect the quality of surgical and postoperative care. The authors of this report examined hospital-level variation in outcomes after inpatient urologic oncology procedures. METHODS: Patients who underwent radical cystectomy, radical nephrectomy, and radical prostatectomy were identified from the Washington State Comprehensive Hospital Abstract Reporting System for the years 2003 through 2007. The postoperative length of stay (LOS) was measured, and LOS that exceeded the 75th percentile was classified as prolonged. The occurrence of Agency for Healthcare Quality patient safety indicators (PSIs), readmissions, and deaths also were measured. Analyses were adjusted for patient age and comorbidity in random effects, multilevel, multivariable models that assessed hospital-level outcomes. RESULTS: The authors identified 853 patients from 37 hospitals who underwent cystectomy, 3018 patients who underwent nephrectomy from 51 hospitals, and 8228 patients who underwent prostatectomy from 51 hospitals. Complications captured by PSIs were rare. Hospital-level variation was most profound for LOS outcomes after nephrectomy and prostatectomy (variance in prolonged LOS, 8.1% and 26.7%, respectively), thromboembolic events after nephrectomy (8% of variance), and mortality after cystectomy (7.1% of variance). CONCLUSIONS: Hospital-level variation confounds the care of urologic cancer patients in the state of Washington. The authors concluded that transparent reporting of surgical outcomes and local quality-improvement initiatives should be considered to ameliorate the observed variation and improve the quality of cystectomy, nephrectomy, and prostatectomy care.

A systematic analysis of a deep mouse epididymal sperm proteome.

Spermatozoa are highly specialized cells that, when mature, are capable of navigating the female reproductive tract and fertilizing an oocyte. The sperm cell is thought to be largely quiescent in terms of transcriptional and translational activity. As a result, once it has left the male reproductive tract, the sperm cell is essentially operating with a static population of proteins. It therefore is theoretically possible to understand the protein networks contained in a sperm cell and to deduce its cellular function capabilities. To this end, we performed a proteomic analysis of mouse sperm isolated from the cauda epididymis and confidently identified 2850 proteins, which to our knowledge is the most comprehensive sperm proteome for any species reported to date. These proteins comprise many complete cellular pathways, including those for energy production via glycolysis, beta-oxidation and oxidative phosphorylation, protein folding and transport, and cell signaling systems. This proteome should prove a useful tool for assembly and testing of protein networks important for sperm function.

Coupling of acceptor-substituted diazo compounds and tertiary thioamides: synthesis of enamino carbonyl compounds and their pharmacological evaluation.

This paper describes the synthesis of enamino carbonyl compounds by the copper(i)-catalyzed coupling of acceptor-substituted diazo compounds and tertiary thioamides. We plan to use this method to synthesize indolizidine (-)-237D analogs to find α6-selective antismoking agents. Therefore, we also performed in silico α6-nAchRs binding studies of selected products. Compounds with low root-mean-square deviation values showed more favorable binding free energies. We also report preliminary pharmacokinetic data on indolizidine (-)-237D and found it to have weak activity at CYP3A4. In addition, as enamino carbonyl compounds are also known for antimicrobial properties, we screened previously reported and new enamino carbonyl compounds for antibacterial, antimicrobial, and antifungal properties. Eleven compounds showed significant antimicrobial activities.

Capillary electrophoretic separation of nanoparticles.

In the present work, CdSe nanocrystals (NCs) synthesized with a trioctylphosphine surface passivation layer were modified using amphiphilic molecules to form a surface bilayer capable of providing stable NCs aqueous solutions. Such modified nanocrystals were used as a test solute in order to analyze new electrophoretic phenomena, by applying a micellar plug as a separation tool for discriminating nanocrystals between micellar and micelle-free zones during electrophoresis. The distribution of NCs between both zones depended on the affinity of nanocrystals towards the micellar zone, and this relies on the kind of surface ligands attached to the NCs, as well as electrophoretic conditions applied. In this case, the NCs that migrated within a micellar zone can be focused using a preconcentration mechanism. By modifying electrophoretic conditions, NCs were forced to migrate outside the micellar zone in the form of a typical CZE peak. In this situation, a two-order difference in separation efficiencies, in terms of theoretical plates, was observed between focused NCs (N ~ 10(7)) and a typical CZE peak for NCs (N ~ 10(5)). By applying the amino-functionalized NCs the preconcentration of NCs, using a micellar plug, was examined, with the conclusion that preconcentration efficiency, in terms of the enhancement factor for peak height (SEF(height)) can be, at least 20. The distribution effect was applied to separate CdSe/ZnS NCs encapsulated in silica, as well as surface-modified with DNA, which allows the estimation of the yield of conjugation of biologically active molecules to a particle surface.

Seminal plasma proteins inhibit in vitro- and cooling-induced capacitation in boar spermatozoa.

Dilute boar seminal plasma (SP) has been shown to inhibit in vitro capacitation and cooling-induced capacitation-like changes in boar spermatozoa, as assessed by the ability of the spermatozoa to undergo an ionophore-induced acrosome reaction. We hypothesised that the protein component of SP is responsible for this effect. To test this hypothesis, varying concentrations of total SP protein or SP proteins fractionated by heparin binding were assayed for their ability to inhibit in vitro capacitation, as well as cooling- and cryopreservation-induced capacitation-like changes. In vitro capacitation and cooling-induced capacitation-like changes were prevented by 10% whole SP, as well as by total proteins extracted from SP at concentrations greater than 500 microg mL(-1). No amount of SP protein was able to prevent cryopreservation-induced capacitation-like changes. Total SP proteins were fractionated based on their heparin-binding properties and the heparin-binding fraction was shown to possess capacitation inhibitory activity at concentrations as low as 250 microg mL(-1). The proteins in the heparin-binding fraction were subjected to mass spectrometry and identified. The predominant proteins were three members of the spermadhesin families, namely AQN-3, AQN-1 and AWN, and SP protein pB1. We conclude that one or more of these heparin-binding SP proteins is able to inhibit in vitro capacitation and cooling-induced capacitation-like changes, but not cryopreservation-induced capacitation-like changes, in boar spermatozoa.

Cellular biophysics during freezing of rat and mouse sperm predicts post-thaw motility.

Though cryopreservation of mouse sperm yields good survival and motility after thawing, cryopreservation of rat sperm remains a challenge. This study was designed to evaluate the biophysics (membrane permeability) of rat in comparison to mouse to better understand the cooling rate response that contributes to cryopreservation success or failure in these two sperm types. In order to extract subzero membrane hydraulic permeability in the presence of ice, a differential scanning calorimeter (DSC) method was used. By analyzing rat and mouse sperm frozen at 5 degrees C/min and 20 degrees C/min, heat release signatures characteristic of each sperm type were obtained and correlated to cellular dehydration. The dehydration response was then fit to a model of cellular water transport (dehydration) by adjusting cell-specific biophysical (membrane hydraulic permeability) parameters L(pg) and E(Lp). A "combined fit" (to 5 degrees C/min and 20 degrees C/min data) for rat sperm in Biggers-Whitten-Whittingham media yielded L(pg) = 0.007 microm min(-1) atm(-1) and E(Lp) = 17.8 kcal/mol, and in egg yolk cryopreservation media yielded L(pg) = 0.005 microm min(-1) atm(-1) and E(Lp) = 14.3 kcal/mol. These parameters, especially the activation energy, were found to be lower than previously published parameters for mouse sperm. In addition, the biophysical responses in mouse and rat sperm were shown to depend on the constituents of the cryopreservation media, in particular egg yolk and glycerol. Using these parameters, optimal cooling rates for cryopreservation were predicted for each sperm based on a criteria of 5%-15% normalized cell water at -30 degrees C during freezing in cryopreservation media. These predicted rates range from 53 degrees C/min to 70 degrees C/min and from 28 degrees C/min to 36 degrees C/min in rat and mouse, respectively. These predictions were validated by comparison to experimentally determined cryopreservation outcomes, in this case based on motility. Maximum motility was obtained with freezing rates between 50 degrees C/min and 80 degrees C/min for rat and at 20 degrees C/min with a sharp drop at 50 degrees C/min for mouse. In summary, DSC experiments on mouse and rat sperm yielded a difference in membrane permeability parameters in the two sperm types that, when implemented in a biophysical model of water transport, reasonably predict different optimal cooling rate outcomes for each sperm after cryopreservation.

Quantification of penicillin G during labor and delivery by capillary electrophoresis.

In this study, a capillary electrophoresis (CE) method was developed as a means to measure levels of penicillin G (PCN G) in Group B Streptococcus (GBS) positive pregnant women during labor and delivery. Volunteers for this developmental study were administered five million units of PCN G at the onset of labor. Urine, blood, and amniotic fluid samples were collected during labor and post delivery. Samples were semi-purified by solid-phase extraction (SPE) using Waters tC18 SepPak 3cc cartridges with a sodium phosphate/methanol step gradient for elution. Capillary electrophoresis or reversed-phase high-performance liquid chromatography (RP-HPLC) with diode-array absorbance detection were used to separate the samples in less than 30 min. Quantification was accomplished by establishing a calibration curve with a linear dynamic range. The tC18 SPE methodology provided substantial sample clean-up with high recovery yields of PCN G ( approximately 90%). It was found that SPE was critical for maintaining the integrity of the separation column when using RP-HPLC, but was not necessary for sample analysis by CE where no stationary phase is present. Quantification results ranged from millimolar concentrations of PCN G in maternal urine to micromolar concentrations in amniotic fluid. Serum and cord blood levels of PCN G were below quantification limits, which is likely due to the prolonged delay in sample collection after antibiotic administration. These results show that CE can serve as a simple and effective means to characterize the pharmacokinetic distribution of PCN G from mother to unborn fetus during labor and delivery. It is anticipated that similar methodologies have the potential to provide a quick, simple, and cost-effective means of monitoring the clinical efficacy of PCN G and other drugs during pregnancy.

Enhanced hot electron lifetimes in quantum wells with inhibited phonon coupling.

Hot electrons established by the absorption of high-energy photons typically thermalize on a picosecond time scale in a semiconductor, dissipating energy via various phonon-mediated relaxation pathways. Here it is shown that a strong hot carrier distribution can be produced using a type-II quantum well structure. In such systems it is shown that the dominant hot carrier thermalization process is limited by the radiative recombination lifetime of electrons with reduced wavefunction overlap with holes. It is proposed that the subsequent reabsorption of acoustic and optical phonons is facilitated by a mismatch in phonon dispersions at the InAs-AlAsSb interface and serves to further stabilize hot electrons in this system. This lengthens the time scale for thermalization to nanoseconds and results in a hot electron distribution with a temperature of 490 K for a quantum well structure under steady-state illumination at room temperature.

Effects of seminal plasma on cooling-induced capacitative changes in boar sperm.

Porcine seminal plasma (SP) has been shown to contain factors that have a decapacitative or capacitation-inhibiting effect on sperm. The objectives of the present study were to compare the capacitative changes observed in cooled sperm with those seen in sperm after in vitro capacitation and to determine whether SP could prevent these changes. Sperm were subjected to incubation or to slow cooling under noncapacitating or capacitating conditions. The effect of SP on protein tyrosine phosphorylation and the ability of the sperm to undergo an acrosome reaction (AR) were determined. Cooled sperm displayed an increased level of tyrosine phosphorylation and a higher percentage of induced AR sperm compared to incubated sperm. The addition of SP inhibited the number of ARs that occurred during incubation and cooling. These results suggest that cooling of sperm augments the capacitative changes in sperm, and that SP contains a factor(s) that effectively prevents these changes.

Structure and function of epididymal protein cysteine-rich secretory protein-1.

Cysteine-rich secretory protein-1 (CRISP-1) is a glycoprotein secreted by the epididymal epithelium. It is a member of a large family of proteins characterized by two conserved domains and a set of 16 conserved cysteine residues. In mammals, CRISP-1 inhibits sperm-egg fusion and can suppress sperm capacitation. The molecular mechanism of action of the mammalian CRISP proteins remains unknown, but certain non-mammalian CRISP proteins can block ion channels. In the rat, CRISP-1 comprises two forms referred to as Proteins D and E. Recent work in our laboratory demonstrates that the D form of CRISP-1 associates transiently with the sperm surface, whereas the E form binds tightly. When the spermatozoa are washed, the E form of CRISP-1 persists on the sperm surface after all D form has dissociated. Cross-linking studies demonstrate different protein-protein interaction patterns for D and E, although no binding partners for either protein have yet been identified. Mass spectrometric analyses revealed a potential post-translational modification on the E form that is not present on the D form. This is the only discernable difference between Proteins D and E, and presumably is responsible for the difference in behavior of these two forms of rat CRISP-1. These studies demonstrate that the more abundant D form interacts with spermatozoa transiently, possibly with a specific receptor on the sperm surface, consistent with a capacitation-suppressing function during sperm transit and storage in the epididymis, and also confirm a tightly bound population of the E form that could act in the female reproductive tract.

Epididymal secreted protein Crisp-1 and sperm function.

Crisp-1 is a member of the cysteine-rich secretory protein family. This family of proteins is characterized by the presence of 16 conserved cysteine residues, the characteristic from which the family name is derived. Members of the Crisp protein family are found in the secretions of the reproductive tract and salivary glands, including venom toxins from several species of snakes and lizards. The Crisp proteins are modular, each containing an amino terminal pathogenesis-related (PR)-like domain and a carboxyl terminal cysteine-rich domain (CRD) connected by a hinge region. Sequence and structural similarities to proteins with known functions suggest that the Crisp family of proteins may act by regulating cellular ion channels. Rat Crisp-1 is synthesized as two distinct isoforms (referred to as Proteins D and E) by the epididymal epithelium and both are secreted into the luminal fluid where they interact with spermatozoa. Our laboratory has correlated Crisp-1 binding to sperm with inhibiting the signaling cascades that initiate capacitation while others have shown that blocking Crisp-1 binding sites on oocytes interferes with sperm-egg fusion. We hypothesize that the D and E populations of rat Crisp-1 have different interactions with sperm that modulate these distinct biological activities. Through tandem mass spectrometry (MS/MS) and monosaccharide composition analyses, we have identified at least one difference between the D and E forms as an additional single O-linked N-acetyl galactosamine on an amino terminal threonine residue in Protein E. This post-translational modification appears to account for the unique 'E' epitope bound by monoclonal antibody 4E9 developed in our laboratory, and may also lead to differential processing and localization of Protein E on sperm, when compared to Protein D. These findings are the first step in distinguishing the molecular basis of the biological activities of the D and E forms of rat Crisp-1. The epididymal-specific expression of Crisp-1, combined with its role in regulation of sperm capacitation and oocyte interaction, make it an attractive target for post-testicular contraceptive development.

Acute effect of vasectomy on the function of the rat epididymal epithelium and vas deferens.

Persistent infertility after apparently successful vasectomy reversal is common. One possible etiology is epididymal epithelial dysfunction resulting in improper sperm maturation after vasectomy reversal. The epididymal epithelium secretes a number of proteins that are thought to be required for the maturation of sperm. Ligation of the vas deferens during vasectomy may affect the synthesis of some of these proteins. In the present study, the function of the epididymal epithelium was assessed at early times after vasectomy (1, 4, and 7 days) by measuring the level of mRNA of 4 secreted proteins: Crisp-1, clusterin, osteopontin, and transferrin. In addition, the site of synthesis of these proteins was determined by immunocytochemistry. The results demonstrated that the expression of Crisp-1 and clusterin, representative epididymal secretory proteins, was largely unaffected by vasectomy. However, osteopontin mRNA increased in the vas deferens in response to vasectomy. Immunocytochemical localization of osteopontin suggested that both infiltrating immune cells and deferential luminal epithelium were responsible for this up-regulation. Transferrin expression was viewed as a marker for immune cells at the site of injury. However, both the caput epididymis and deferential epithelia were found to express transferrin, in addition to immune cells. In conclusion, there appear to be only minor changes in expression of genes encoding epididymal secretory proteins acutely after vasectomy, but, not surprisingly, there was evidence of an inflammatory response after vasectomy.

Travel Burden and the Direct Medical Costs of Urologic Surgery.

Background: Increased surgical volume is associated with better patient outcomes and shorter lengths of hospitalization. As a consequence, traveling to receive care from a high volume provider may be associated with better outcomes. However, travel may also be associated with a decision by the healthcare provider to increase the length of stay due to a decreased ability to return to the primary hospital should complications arise. Thus, research is needed to understand the relationship between the distance a patient must travel and their outcomes following urologic surgery. Objective: The purpose of this study was to determine whether the distance a patient travels to receive urologic surgery is associated with their length of hospital stay and direct medical hospitalization costs. Methods: This was a retrospective observational cohort study of 12 106 patients over 50 years of age undergoing transurethral resection of the prostate (TURP), radical prostatectomy (RP) or radical cystectomy (RC) in Washington State hospitals between 2009 and 2013. Distance traveled was determined by calculating the linear distance between zip code centroids of patient residence and the hospital performing their procedure. Patients were sorted into four groups classified by distance traveled (≤5 miles, 6-20 miles, 21-50 miles and ≥51 miles) and cost calculated using a charges-to-reimbursement ratio for each hospital. Statistical significance was determined using a Kruskal-Wallis test. Results: Patients traveling greater distances had significantly lower median medical costs compared with patients who lived closer to the hospitals where they underwent TURP and RP (TURP: ≤5 miles, $6243 and ≥51 miles, $5105, p≤0.001; RP: ≤5 miles, $12 407 and ≥51 miles, $11 882, p≤0.001), whereas there was no significant difference for patients undergoing RC (≤5 miles, $27 554 and ≥51 miles, $26 761, p=0.17). Likewise, patients traveling greater distances had significantly lower median lengths of hospitalization for TURP and RP (TURP: p≤0.001, RP: p≤0.001), while there was no difference for RC (p=0.50). Conclusions: Patient travel burden does appear to play a role in cost and length of hospital stay for select urologic procedures with variable levels of morbidity and recovery time. Although these findings are statistically significant, the magnitude of the effect is small.

Hospital-level Variation in the Quality of Benign Inpatient Urologic Surgery.

OBJECTIVE: To examine hospital-level variation in outcomes following benign urologic surgeries given that hospital-level variation in surgical outcomes can portend quality and appropriateness of care concerns and identify quality improvement opportunities in perioperative care. MATERIALS AND METHODS: Using the Washington State Comprehensive Hospital Abstract Reporting System, we identified patients who underwent transurethral resection of the prostate (TURP), percutaneous nephrostolithotomy (PCNL), and pyeloplasty from 2003 to 2008. We classified prolonged postoperative length of stay (LOS) as that exceeding the 75th percentile, and we measured the rate of Agency for Healthcare Quality Patient Safety Indicators, readmissions, and death. We calculated hospital-specific observed-to-expected event rates using random effects multilevel multivariable models adjusted for age and comorbidity. RESULTS: We identified 6699 TURP patients at 54 hospitals, 2541 PCNL patients at 45 hospitals, and 584 pyeloplasty patients at 36 hospitals. Complication rates were highest after PCNL (22.9% prolonged LOS vs 17.3% for TURP and 13.9% for pyeloplasty, P < .001; 3.4% 90-day mortality vs 0.6% for TURP and 0% for pyeloplasty). Hospital-level variation was most substantial for LOS after TURP and pyeloplasty (8.1% and 14.3% of variance in prolonged LOS, respectively). CONCLUSION: Hospital-level variation is common after benign inpatient urologic surgeries and may relate to difference in perioperative provider practice patterns. The morbidity of PCNL in this study was higher than expected and merits further investigation.

Analysis of the human sperm proteome.

As part of our effort to identify putative protein targets for the development of male contraceptives, we performed an in-depth proteomic analysis of human sperm by liquid chromatography and tandem mass spectrometry. Motile sperm were collected from a single fertile individual and fractionated into detergent-soluble and detergent-insoluble fractions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation of these fractions, followed by manual cutting of the gel, yielded 35 gel sections for each fraction to include proteins across the full range of electrophoretic mobility. Proteomic analysis of these gel sections identified more than 1,760 proteins with high confidence, with 1,350 proteins identified in the soluble fraction, 719 identified in the insoluble fraction, and 309 identified in both fractions. This characterization of the human sperm proteome provides a high-resolution, physiologically relevant index of the proteins that comprise human sperm.

Effect of pH on radiation-induced p53 expression.

PURPOSE: In most tumors, the intratumor environment is acidic. The purpose of this study was to elucidate the effect of acidic extracellular environment on the radiation-induced expression of p53 and related molecular signals. METHODS AND MATERIALS: Cultured RKO.C human colorectal cancer cells carrying wild-type p53 were used. Cells grown in pH 7.5 medium or pH 6.6 medium were irradiated with gamma-rays, and the expression of p53 and p53 mRNA, as well as the degradation rate of the molecules, was determined. The transcriptional activity for p53 was investigated using cells transfected with a p53 reporter construct. The expression of Mdm2 and the phosphorylation of p53, essential factors for p53 degradation, were also investigated. RESULTS: The pH 6.6 environment prolonged the radiation-induced expression of p53 and p53 mRNA. The radiation-induced increase in transcriptional activity of p53 lasted longer in pH 6.6 medium than in pH 7.5 medium. The degradation of p53 was delayed at pH 6.6. The radiation-induced expression of Mdm2 was markedly suppressed, whereas the phosphorylation of p53 was markedly increased after irradiation in pH 6.6 medium. CONCLUSION: Acidic environment significantly enhances the radiation-induced expression of p53, partly by increasing the formation of p53 and also partly by slowing down the degradation of p53 through inhibiting p53-Mdm2 complex formation. The potential implication of acidic intratumor microenvironment for the response of tumors to radiotherapy remains to be elucidated.

Assessing pH and oxygenation in cryotherapy-induced cytotoxicity and tissue response to freezing.

The microenvironmental pH and oxygenation is known to influence tumor cell response to heat, radiation, photodynamic and even chemotherapy. We have studied the previously untested influence of acidity and hypoxia on tumor and endothelial cell sensitivity to freezing. In addition, we have measured changes in oxygenation in vivo in murine FSaII fibrosarcomas after freeze injury. A low pH or low oxygenation environment was found to increase the sensitivity of tumor and endothelial cells to freezing at -20 degrees C or -40 degrees C in vitro. However, low pH and low oxygenation combined did not further increase cryosensitivity of the cells. In vivo, tumor oxygenation after freeze injury was studied immediately or 1-3 days after a standard freezing protocol was applied to FSaII tumors ranging from 250-500 mm3 grown in the rear-limb of C3H mice. Tumor oxygenation at the edge of the iceball was found to transiently increase 1-2 hours after freezing. At 1-3 days after freezing, a treatment that delayed FSaII tumor growth by approximately 1.5-fold, the mean tumor oxygenation was significantly increased by up to 2.5-fold from a control level of 5 mmHg partial pressure of oxygen (pO2), especially at the periphery of the tumor. We conclude that manipulation of pH or oxygenation has potential to increase the anti-tumor effects of minimally invasive cryosurgical techniques. Furthermore, the dynamic changes in oxygenation after freeze injury in vivo suggests value in combining cryotherapy with treatments dependent on oxygenation levels. Ultimately, these may be routes to more reliable treatment response with fewer recurrences.

Mechanical property characterization of mouse zona pellucida.

Previous intracytoplasmic sperm injection (ICSI) studies have indicated significant variation in ICSI success rates among different species. In mouse ICSI, the zona pellucida (ZP) undergoes a "hardening" process at fertilization in order to prevent subsequent sperm from penetrating. There have been few studies investigating changes in the mechanical properties of mouse ZP post fertilization. To characterize mouse ZP mechanical properties and quantitate the mechanical property differences of the ZP before and after fertilization, a microelectromechanical systems-based multiaxis cellular force sensor has been developed. A microrobotic cell manipulation system employing the multiaxis cellular force sensor is used to conduct mouse ZP force sensing, establishing a quantitative relationship between applied forces and biomembrane structural deformations on both mouse oocytes and embryos. An analytical biomembrane elastic model is constructed to describe biomembrane mechanical properties. The characterized elastic modulus of embryos is 2.3 times that of oocytes, and the measured forces for puncturing embryo ZP are 1.7 times those for oocyte ZP. The technique and model presented in this paper can be applied to investigations into the mechanical properties of other biomembranes, such as the plasma membrane of oocytes or other cell types.