Optimizing multiphoton fluorescence microscopy light collection from living tissue by noncontact total emission detection (epiTED).

A benefit of multiphoton fluorescence microscopy is the inherent optical sectioning that occurs during excitation at the diffraction-limited spot. The scanned collection of fluorescence emission is incoherent; that is, no real image needs to be formed on the detector plane. The nearly isotropic emission of fluorescence excited at the focal spot allows for new detection schemes that efficiently funnel all attainable photons to detector(s). We previously showed [Combs, C.A., et al. (2007) Optimization of multiphoton excitation microscopy by total emission detection using a parabolic light reflector. J. Microsc. 228, 330-337] that parabolic mirrors and condensers could be combined to collect the totality of solid angle around the excitation spot for tissue blocks, leading to ∼8-fold signal gain. Using a similar approach, we have developed an in vivo total emission detection (epiTED) instrument modified to make noncontact images from outside of living tissue. Simulations suggest that a ∼4-fold enhancement may be possible (much larger with lower NA objectives than the 0.95 NA used here) with this approach, depending on objective characteristics, imaging depth and the characteristics of the sample being imaged. In our initial prototype, 2-fold improvements were demonstrated in the mouse brain and skeletal muscle as well as the rat kidney, using a variety of fluorophores and no compromise of spatial resolution. These results show this epiTED prototype effectively doubles emission signal in vivo; thus, it will maintain the image signal-to-noise ratio at two times the scan rate or enable full scan rate at approximately 30% reduced laser power (to minimize photo-damage).

1α,24(S)(OH)2D2 normalizes bone morphology and serum parathyroid hormone without hypercalcemia in 25-hydroxyvitamin D-1-hydroxylase (CYP27B1)-deficient mice, an animal model of vitamin D deficiency with secondary hyperparathyroidism.

BACKGROUND: Vitamin D compounds are effective in managing elevated PTH levels in secondary hyperparathyroidism (SHPT) of renal failure. However, undesired increases in serum calcium and phosphorus associated with compounds such as calcitriol [1,25(OH)2D3] has prompted a search for compounds with improved safety profiles. 1alpha,24(S)(OH)2D2 (1,24(OH)2D2) is a vitamin D2 metabolite with low calcium-mo bilizing activity in vivo. We studied the efficacy of 1,24(OH)2D2 in mice lacking the CYP27B1 enzyme [25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-OHase)], a novel vitamin D deficiency model with SHPT. MATERIALS AND METHODS: 1alpha-OHase-deficient (-/-) mice and normal (+/-) heterozygous littermates re ceived 1,24(OH)2D2 (100, 300, 1000, and 3000 pg/g/day) or 1,25(OH)2D3 (30, 300, and 500 pg/g/day) for 5 weeks via daily sc injection. Control groups received vehicle. RESULTS: Vehicle-treated 1alpha-OHase-deficient mice were hypocalcemic and had greatly elevated serum PTH. 1,24(OH)2D2 at doses above 300 pg/g/day normalized serum calcium, serum PTH, bone growth plate morphology, and other bone parameters. No hy percalcemia was observed at any dose of 1,24(OH)2D2 in normal or 1alpha-OHase-deficient animals. In contrast, 1,25(OH)2D3 at only 30 pg/g/day normalized calcemia, serum PTH, and bone parameters, but at higher doses completely suppressed PTH and caused hypercalcemia in both 1alpha-OHase-deficient and normal mice. Treatment with 500 pg/g/day of 1,25(OH)2D3 also induced osteomalacia in normal animals. CONCLUSION: 1,25(OH)2D3 was maximally active at 10-fold lower doses than 1,24(OH)2D2, but induced hypercalcemia and osteomalacia at high doses. 1,24(OH)2D2 normalized serum calcium, serum PTH, and bone histomorphometry without hypercalcemia in 1alpha-OHase-deficient mice with SHPT.

Growth inhibition of cancer cells by an active metabolite of a novel vitamin D prodrug.

Active vitamin D compounds have been developed that maintain antiproliferative properties with low calcemic activity. BCI-210, a novel vitamin D pro-drug developed in our laboratory, is activated through side chain hydroxylation and possesses lower calcemic activity than calcitriol. The human hepatoma cell line (HepG2) was used to produce an active metabolite, which was characterized and identified as 27-hydroxy-BCI-210. We compared the ability of 27-OH-BCI-210 with calcitriol to inhibit proliferation of prostate (LNCaP), and breast (MCF-7) cancer cells. Cells were plated in multi-well plates and incubated with vehicle or vitamin D compounds for 6 days, after which the cell numbers were determined by a colorimetric assay. 27-OH-BCI-210 produced a dose-dependent growth inhibition, although a concentration five-fold greater than calcitriol was required to produce equivalent inhibition. We also examined the antiproliferative activity of 27-OH-BCI-210 in combination with chemotherapeutic drugs. With genistein and doxorubicin, 27-OH-BCI-210 produced synergistic inhibition of proliferation of LNCaP and MCF-7 cells. These synergistic interactions suggest the potential clinical utility of 27-OH-BCI-210 in the treatment of prostate and breast tumors.

Pamidronate and 1,24(S)-dihydroxyvitamin D2 synergistically inhibit the growth of myeloma, breast and prostate cancer cells.

BACKGROUND: Bisphosphonates have proven to be effective in the management of multiple myeloma and bone metastases secondary to breast and prostate carcinoma. Vitamin D compounds are important modulators of cellular proliferation and differentiation. 1,24(S)-dihydroxyvitamin D2 [1,24(OH)2D2] is a naturally occurring active vitamin D compound with high antiproliferative activity and low calcemic response. MATERIALS AND METHODS: We examined the antiproliferative effects of 1,24(OH)2D2 in combination with the bisphosphonate pamidronate on multiple myeloma (H929), prostate (LNCaP) and breast (MCF-7) cancer cell lines. Drug-drug interactions were analyzed using the median-effect/isobologram method to characterize the interactions as synergistic, additive, or antagonistic. RESULTS: Pamidronate and 1,24(OH)2D2 were found independently to inhibit cancer cell growth in a dose-dependent manner. Combinations of these compounds produced marked synergistic growth-inhibitory effects at several clinically relevant concentrations. CONCLUSION: Combined dosing of pamidronate and 1,24(OH)2D2 may have therapeutic value for the treatment of multiple myeloma, prostate and breast cancers.

Combination study of 1,24(S)-dihydroxyvitamin D2 and chemotherapeutic agents on human breast and prostate cancer cell lines.

BACKGROUND: Vitamin D compounds are important modulators of cellular proliferation and differentiation, with potential utility as anticancer drugs. 1,24(S)-Dihydroxyvitamin D2 [1,24(OH)2D2] is a naturally occurring active vitamin D compound with low calcemic activity. MATERIALS AND METHODS: We evaluated the growth inhibitory effects of 1,24(OH)2D2 on LNCaP prostate cancer and MCF-7 breast cancer cells. 1,24(OH)2D2 was evaluated alone and in paired combination with nine chemotherapeutic agents. Drug interactions were analyzed using the median-effect/isobologram method. Combination index values were used to characterize the interactions as synergistic, additive, or antagonistic. RESULTS: In MCF-7 cells, 1,24(OH)2D2 produced synergistic effects with doxonrubicin and cisplatin and additive effects with busulfan, etoposide, tamaxifen, 5-fluorouracil and carboplatin. In LNCaP cells, 1,24(OH)2D2 produced a synergistic effect with carboplatin and additive effects with doxorubicin, busulfan, paclitaxel and etoposide. CONCLUSION: We conclude that 1,24(OH)2D2 may have therapeutic value in the treatment of prostate and breast cancers, alone or in combination with chemotherapeutic agents.

Selectivity and sensitivity of intramuscular and transcutaneous electromyography electrodes.

We evaluated the differences in selectivity and sensitivity of intramuscular fine-wire electrodes and transcutaneous electrodes in detecting dynamic electromyography (EMG) signals from extensor digitorum (EDC) and extensor carpi radialis (ECR) muscles during isolated EDC and ECR contractions in two able-bodied subjects. Intramuscular fine-wire electrodes differentiated EDC and ECR EMG activities better than transcutaneous electrodes, and intramuscular fine-wire electrodes recorded higher amplitude signals than transcutaneous electrodes. Data suggest that intramuscular fine-wire electrodes are more selective and sensitive than transcutaneous electrodes in detecting EMG signals from adjacent forearm muscles.

Intramuscular electromyographically controlled neuromuscular electrical stimulation for ankle dorsiflexion recovery in chronic hemiplegia.

We report three cases of survivors of chronic stroke treated with active repetitive movement training of the paretic ankle dorsiflexors mediated by intramuscular electromyographically controlled neuromuscular electrical stimulation (NMES). These case reports demonstrate the feasibility of using intramuscular electromyographically controlled NMES for facilitating ankle dorsiflexion recovery among survivors chronic stroke with moderate hemiplegia. Relevant issues for clinical implementation and future investigations are reviewed.

Maltreatment and disabilities: a population-based epidemiological study.

OBJECTIVES: To assess the prevalence of abuse and neglect among a population of children identified as a function of an existing disability, relate specific types of disabilities to specific types of abuse, and to determine the effect of abuse and neglect on academic achievement and attendance rates for children with and without disabilities. METHOD: An electronic merger of school records with Central Registry, Foster Care Review Board, and police databases was followed by a detailed record review of the circumstances of maltreatment. RESULTS: Analyses of the circumstances of maltreatment and the presence of disabilities established a 9% prevalence rate of maltreatment for nondisabled children and a 31% prevalence rate for the disabled children. Thus, the study established a significant association between the presence of an educationally relevant disability and maltreatment. CONCLUSIONS: Children with disabilities are 3.4 times more likely to be maltreated than nondisabled peers. School professionals need to be cognizant of the high base rate of maltreatment among the children they serve. Disability status needs to be considered in national incidence studies of maltreatment.

The prevalence of disabilities and maltreatment among runaway children.

OBJECTIVE: This research was conducted to determine the prevalence of disabilities among abused and nonabused runaways within a hospital population (Study 1) and community school population (Study 2) and to identify any associations between disability, maltreatment, family stress factors, academic achievement, school attendance, domestic violence and runaway status. METHOD: Descriptive information was collected for maltreated and nonmaltreated runaways from hospital (N = 39,352; 255 runaways) and school (N = 40,211; 562 runaways) populations including: disability status, type of maltreatment, family stress factors, record of domestic violence in the family, academic achievement and attendance. RESULTS: The prevalence rate of disabilities among the maltreated runaways was 83.1% and 47% among the nonmaltreated runaways in the hospital sample and 34% and 17%, respectively, in the school sample. Children and youth with disabilities were at increased risk to become runaways in both populations. The presence of maltreatment significantly increased the association between running away and disability status. Children with behavior disorders, mental retardation, and some type of communication disorder were significantly more likely to run away than children with other disabilities. Among the maltreated runaways with and without disabilities, physical abuse and sexual abuse were significantly associated with running away. Records of domestic violence were more prevalent in the families of runaways with behavior disorders and no diagnosed disability. Lower academic achievement, poor school attendance, and more family stress factors were associated with maltreatment, disability and runaway status. CONCLUSIONS: Children and youth with disabilities are unidentified and unrecognized among runaways. Professionals working with runaways and their families need to be cognizant of the special needs of the population, particularly with respect to behavior disorders, communication disabilities, and mental retardation and reconsider current policy to routinely reunite runaways with their families when running away was precipitated by traumatagenic factors within the family.

Musical backgrounds, listening habits, and aesthetic enjoyment of adult cochlear implant recipients.

This paper describes the listening habits and musical enjoyment of postlingually deafened adults who use cochlear implants. Sixty-five implant recipients (35 females, 30 males) participated in a survey containing questions about musical background, prior involvement in music, and audiologic success with the implant in various listening circumstances. Responses were correlated with measures of cognition and speech recognition. Sixty-seven implant recipients completed daily diaries (7 consecutive days) in which they reported hours spent in specific music activities. Results indicate a wide range of success with music. In general, people enjoy music less postimplantation than prior to hearing loss. Musical enjoyment is influenced by the listening environment (e.g., a quiet room) and features of the music.

Intrinsic and extrinsic contributions to the passive moment at the metacarpophalangeal joint.

The purpose of this investigation was to determine whether the passive range of motion at the finger joints is restricted more by intrinsic tissues (cross a single joint) or by extrinsic tissues (cross multiple joints). The passive moment at the metacarpophalangeal (MP) joint of the index finger was modeled as the sum of intrinsic and extrinsic components. The intrinsic component was modeled only as a function of MP joint angle. The extrinsic component was modeled as a function of MP joint angle and wrist angle. With the wrist fixed in seven different positions the passive moment at the MP joint of eight subjects was recorded as the finger was rotated through its range at a constant rate. The moment-angle data were fit by the model and the extrinsic and intrinsic components were calculated for a range of MP joint angles and wrist positions. With the MP joint near its extension limit, the median percent extrinsic contribution was 94% with the wrist extended 60 degrees and 14% with the wrist flexed 60 degrees. These percentages were 40 and 88%, respectively, with the MP joint near its flexion limit. Our findings indicate that at most wrist angles the extrinsic tissues offer greater restraint at the limits of MP joint extension and flexion than the intrinsic tissues. The intrinsic tissues predominate when the wrist is flexed or extended enough to slacken the extrinsic tissues. Additional characteristics of intrinsic and extrinsic tissues can be deduced by examining the parameter values calculated by the model.

Nanosecond dynamics of the single tryptophan reveals multi-state equilibrium unfolding of protein GB1.

Unfolding of the immunoglobulin binding domain B1 of streptococcal protein G (GB1) was induced by guanidine hydrochloride (GdnHCl) and studied by circular dichroism, steady-state, and time-resolved fluorescence spectroscopy. The fluorescence methods employed the single tryptophan residue of GB1 as an intrinsic reporter. While the transitions monitored by circular dichroism and steady-state fluorescence coincided with each other, the transitions followed by dynamic fluorescence were markedly different. Specifically, fluorescence anisotropy data showed that a relaxation spectrum of tryptophan contained a slow motion with relaxation times of 9 ns in the native state and 4 ns in the unfolded state in 6 M GdnHCl. At intermediate GdnHCl concentrations of 3.8-4.2 M, however, the slow relaxation time increased to 18 ns. The fast nanosecond motion had an average time of 0.8 ns and showed no dependence on the formation of native structure. Overall, dynamic fluorescence revealed two preliminary stages in GB1 folding, which are equated with the formation of local structure in the beta(3)-strand hairpin and the initial collapse. Both stages exist without alpha-helix formation, i. e., before the appearance of any ordered secondary structure detectable by circular dichroism. Another stage in GB1 folding might exist at very low ( approximately 1 M) GdnHCl concentrations.

Comparison of the relative effects of 1,24-dihydroxyvitamin D(2) [1, 24-(OH)(2)D(2)], 1,24-dihydroxyvitamin D(3) [1,24-(OH)(2)D(3)], and 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] on selected vitamin D-regulated events in the rat.

The present experiments were conducted to compare the relative hypercalciuric and hypercalcemic activities of 1,24-dihydroxyvitamin D(2) [1,24-(OH)(2)D(2)], 1,24-dihydroxyvitamin D(3) [1, 24-(OH)(2)D(3)], and 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] in 7-week-old rats. The rats were dosed orally with each sterol for 7 days at a rate of 1 ng/g body weight/day. We also monitored the effect of the three compounds on the induction of mRNA for CaATPase and for 25-hydroxyvitamin D-24-hydroxylase in the kidney and intestine, on plasma vitamin D metabolite levels, and on the capacity to evoke modification in the vitamin D receptor/retinoic acid X receptor (VDR/RXR) heterodimer conformation. Plasma calcium was elevated in the rats treated with 1,24-(OH)(2)D(3) and 1, 25-(OH)(2)D(3), but not in the 1,24-(OH)(2)D(2)-dosed rats. Urinary calcium was elevated significantly (relative to controls) in all groups. The order of hypercalciuric activity was 1,25-(OH)(2)D(3) >/= 1,24-(OH)(2)D(3) >/= 1,24-(OH)(2)D(2) > control. Duodenal plasma membrane calcium ATPase (PMCA) mRNA was elevated to a similar extent in all groups relative to controls. Duodenal 24-hydroxylase mRNA was elevated in all groups relative to controls; however, the elevations were significantly higher in the 1,24-(OH)(2)D(3) and 1, 25-(OH)(2)D(3) groups compared with the 1,24-(OH)(2)D(2) group. Kidney 24-hydroxylase also was elevated significantly in the 1, 24-(OH)(2)D(3)- and 1,25-(OH)(2)D(3)-treated rats but not in the 1, 24-(OH)(2)D(2)-treated rats. Recombinant human vitamin D receptor (hVDR) extracts were incubated with saturating concentrations of 1, 24-(OH)(2)D(2), 1,24-(OH)(2)D(3), and 1,25-(OH)(2)D(3) and subsequently analyzed by electrophoretic mobility shift assay (EMSA). Overall binding was comparable for all metabolites; however, the 1, 24-(OH)(2)D(2) complex exhibited distinctly altered mobility relative to 1,24-(OH)(2)D(3) and 1,25-(OH)(2)D(3), suggestive of an effect on hVDR/hRXR conformation. These data suggest that 1, 24-(OH)(2)D(2) is not as potent as either of the vitamin D(3) sterols at affecting hypercalcemia or hypercalciuria in young growing rats; however, 1,24-(OH)(2)D(2) can evoke other biological responses similar to the vitamin D(3) sterols. These different responses may be related to the alterations in conformation state of the hVDR/hRXR heterodimer.

The veridicality of punitive childhood experiences reported by adolescents and young adults.

OBJECTIVE: The primary goal of the present research was to determine whether retrospective reports of childhood disciplinary experiences and perceptions of that discipline correspond to actual childhood events and whether the accuracy of that report was influenced by the affective state of the respondent. METHOD: Eighty-three adolescent and young adult males completed a retrospective measure of physical child maltreatment, Assessing Environments (AEIII), and the Brief Symptom Inventory (BSI). As children the participants had been observed naturalistically in their homes interacting with their parents an average of 10 years earlier. RESULTS: Analyses were consistent with the hypothesis that both current mood and actual observations of parent-child interactions during childhood predict self-reported recollections of childhood maltreatment by one's parents. Further the veridicality of such recollections appears to depend upon the objective specificity versus the perceptive nature of the questions used to elicit the recollections. CONCLUSIONS: The findings suggest that assessment instruments suitable for obtaining information regarding earlier childhood victimization must utilize behaviorally specific items. Thus, items that are either global or intimate a normative comparison should be avoided.

Does intraoperative hetastarch administration increase blood loss and transfusion requirements after cardiac surgery?

UNLABELLED: Hetastarch is used for intravascular volume expansion in cardiac surgery. Studies show conflicting effects of intraoperative hetastarch administration on postoperative bleeding. Hetastarch was routinely used for volume expansion during cardiovascular surgeries at our institution until its use was discontinued intraoperatively. We performed a retrospective chart review on patients undergoing primary coronary artery bypass grafting, valve repair or replacement requiring cardiopulmonary bypass (n = 444), 234 of which received intraoperative hetastarch and 210 did not. There was no difference in demographics, cardiac surgery, or cardiopulmonary bypass duration between the two groups. Blood loss for 0-4 h postoperatively was 377 +/- 244 mL in the group not receiving hetastarch compared with 515 +/- 336 mL in the group that received hetastarch (P < 0.001). For 0-24 h postoperatively, blood loss was 923 +/- 473 mL versus 1,283 +/- 686 mL in the absence and presence of hetastarch, respectively (P < 0.001). Allogeneic transfusion requirements (cryoprecipitate, fresh frozen plasma, and platelets) were larger in the hetastarch group (all P < 0.001). Nearly all (99%) patients in the hetastarch group received less than the manufacturer's recommended dose (20 mL/kg) of hetastarch. IMPLICATIONS: Our large retrospective study suggests that intraoperative use of hetastarch in primary cardiac surgery with cardiopulmonary bypass may increase bleeding and transfusion requirements. A large prospective study is needed to determine if intraoperative administration of hetastarch should be avoided during cardiovascular surgery.

Nanosecond dynamics of tryptophans in different conformational states of apomyoglobin proteins.

Fluorescence anisotropy kinetics were employed to quantify the nanosecond mobility of tryptophan residues in different conformational states (native, molten globule, unfolded) of apomyoglobins. Of particular interest is the similarity between the fluorescence anisotropy decays of tryptophans in the native and molten globule states. We find that, in these compact states, tryptophan residues rotate rapidly within a cone of semiangle 22-25 degrees and a correlation time of 0.5 ns, in addition to rotating together with the whole protein with a correlation time of 7-11 ns. The similar nanosecond dynamics of tryptophan residues in both states suggests that the conformation changes that distinguish the molten globule and native states of apomyoglobins originate from either subtle, slow rearrangements or fast changes distant from these tryptophans.