Improved Inhibitory and Absorption, Distribution, Metabolism, Excretion, and Toxicology (ADMET) Properties of Blebbistatin Derivatives Indicate That Blebbistatin Scaffold Is Ideal for drug Development Targeting Myosin-2.

Blebbistatin, para-nitroblebbistatin (NBleb), and para-aminoblebbistatin (AmBleb) are highly useful tool compounds as they selectively inhibit the ATPase activity of myosin-2 family proteins. Despite the medical importance of the myosin-2 family as drug targets, chemical optimization has not yet provided a promising lead for drug development because previous structure-activity-relationship studies were limited to a single myosin-2 isoform. Here we evaluated the potential of blebbistatin scaffold for drug development and found that D-ring substitutions can fine-tune isoform specificity, absorption-distribution-metabolism-excretion, and toxicological properties. We defined the inhibitory properties of NBleb and AmBleb on seven different myosin-2 isoforms, which revealed an unexpected potential for isoform specific inhibition. We also found that NBleb metabolizes six times slower than blebbistatin and AmBleb in rats, whereas AmBleb metabolizes two times slower than blebbistatin and NBleb in human, and that AmBleb accumulates in muscle tissues. Moreover, mutagenicity was also greatly reduced in case of AmBleb. These results demonstrate that small substitutions have beneficial functional and pharmacological consequences, which highlight the potential of the blebbistatin scaffold for drug development targeting myosin-2 family proteins and delineate a route for defining the chemical properties of further derivatives to be developed. SIGNIFICANCE STATEMENT: Small substitutions on the blebbistatin scaffold have beneficial functional and pharmacological consequences, highlighting their potential in drug development targeting myosin-2 family proteins.

A Landauer Formula for Bioelectronic Applications.

Recent electronic transport experiments using metallic contacts attached to proteins identified some "stylized facts", which contradict conventional wisdom that increasing either the spatial distance between the electrodes or the temperature suppresses conductance exponentially. These include nearly temperature-independent conductance over the protein in the 30 to 300 K range, distance-independent conductance within a single protein in the 1 to 10 nm range and an anomalously large conductance in the 0.1 to 10 nS range. In this paper, we develop a generalization of the low temperature Landauer formula, which can account for the joint effects of tunneling and decoherence and can explain these new experimental findings. We use novel approximations, which greatly simplify the mathematical treatment and allow us to calculate the conductance in terms of a handful macroscopic parameters, instead of the myriads of microscopic parameters describing the details of an atomic level quantum chemical computation. The new approach makes it possible to get predictions for the outcomes of new experiments without relying solely on high performance computing and can distinguish important and unimportant details of the protein structures from the point of view of transport properties.

Knowledge, actual and potential use of HIV self-sampling testing kits among MSM recruited in eight European countries.

AIM: To describe the knowledge as well as current and potential use of self-sampling kits among men who have sex with men (MSM) and to analyse their preferred biological sample and result communication method. METHODS: We analyse data of MSM of HIV negative or unknown serostatus from an online survey conducted in eight countries (Belgium, Denmark, Germany, Greece, Portugal, Romania, Slovenia and Spain) between April and December 2016. It was advertised mainly in gay dating websites. We conduct a descriptive analysis of the main characteristics of the participants, and present data on indicators of knowledge, use and potential use of HIV self-sampling as well as their preferences regarding blood or saliva sample and face or non-face-to-face result communication by country of residence. RESULTS: A total of 8.226 participants of HIV negative or unknown serostatus were included in the analysis. Overall, 25.5% of participants knew about self-sampling (range: 18.8-47.2%) and 1.1% had used it in the past (range: 0.3-8.9%). Potential use was high, with 66.6% of all participants reporting that they would have already used it if available in the past (range: 62.1-82.1%). Most (78.6%) reported that they would prefer using a blood-based kit, and receiving the result of the test through a non-face-to-face-method (70.8%), even in the case of receiving a reactive result. CONCLUSION: The high potential use reported by MSM recruited in eight different European countries suggests that self-sampling kits are a highly acceptable testing methodology that could contribute to the promotion of HIV testing in this population.

Morphological evidence for enhanced kisspeptin and neurokinin B signaling in the infundibular nucleus of the aging man.

Peptidergic neurons synthesizing kisspeptin (KP) and neurokinin B (NKB) in the hypothalamic infundibular nucleus have been implicated in negative sex steroid feedback to GnRH neurons. In laboratory rodents, testosterone decreases KP and NKB expression in this region. In the present study, we addressed the hypothesis that the weakening of this inhibitory testosterone feedback in elderly men coincides with enhanced KP and NKB signaling in the infundibular nucleus. This central hypothesis was tested in a series of immunohistochemical studies on hypothalamic sections of male human individuals that were divided into arbitrary "young" (21-49 yr, n = 11) and "aged" (50-67 yr, n = 9) groups. Quantitative immunohistochemical experiments established that the regional densities of NKB-immunoreactive (IR) perikarya and fibers, and the incidence of afferent contacts they formed onto GnRH neurons, exceeded several times those of the KP-IR elements. Robust aging-dependent enhancements were identified in the regional densities of KP-IR perikarya and fibers and the incidence of afferent contacts they established onto GnRH neurons. The abundance of NKB-IR perikarya, fibers, and axonal appositions to GnRH neurons also increased with age, albeit to lower extents. In dual-immunofluorescent studies, the incidence of KP-IR NKB perikarya increased from 36% in young to 68% in aged men. Collectively, these immunohistochemical data suggest an aging-related robust enhancement in central KP signaling and a moderate enhancement in central NKB signaling. These changes are compatible with a reduced testosterone negative feedback to KP and NKB neurons. The heavier KP and NKB inputs to GnRH neurons in aged, compared with young, men may play a role in the enhanced central stimulation of the reproductive axis. It requires clarification to what extent the enhanced KP and NKB signaling upstream from GnRH neurons is an adaptive response to hypogonadism or, alternatively, a consequence of a decline in the androgen sensitivity of KP and NKB neurons.

Low degree of overlap between kisspeptin, neurokinin B, and dynorphin immunoreactivities in the infundibular nucleus of young male human subjects challenges the KNDy neuron concept.

Previous immunohistochemical and in situ hybridization studies of sheep, goats, and rodents indicated that kisspeptin (KP), neurokinin B (NKB), and dynorphin A (DYN) are extensively colocalized in the hypothalamic arcuate nucleus, thus providing a basis for the KP/NKB/DYN (KNDy) neuron concept; in both sexes, KNDy neuropeptides have been implicated in the generation of GnRH neurosecretory pulses and in the negative feedback effects of sexual steroids to the reproductive axis. To test the validity and limitations of the KNDy neuron concept in the human, we carried out the comparative immunohistochemical analysis of the three neuropeptides in the infundibular nucleus (Inf; also known as arcuate nucleus) and stalk of young male human individuals (<37 yr). Results of quantitative immunohistochemical experiments established that the regional densities of NKB immunoreactive (IR) perikarya and fibers, and the incidence of afferent contacts they formed onto GnRH neurons, were about 5 times as high as those of the KP-IR elements. Dual-immunofluorescent studies confirmed that considerable subsets of the NKB-IR and KP-IR cell bodies and fibers are separate, and only about 33% of NKB-IR perikarya and 75% of KP-IR perikarya were dual labeled. Furthermore, very few DYN-IR cell bodies could be visualized in the Inf. DYN-IR fibers were also rare and, with few exceptions, distinct from the KP-IR fibers. The abundance and colocalization patterns of the three immunoreactivities showed similar trends in the infundibular stalk around portal blood vessels. Together these results indicate that most NKB neurons in the Inf do not synthesize detectable amounts of KP and DYN in young male human individuals. These data call for a critical use of the KNDy neuron terminology when referring to the putative pulse generator system of the mediobasal hypothalamus. We conclude that the functional importance of these three neuropeptides in reproductive regulation considerably varies among species, between sexes, and at different ages.

Sexual dimorphism of kisspeptin and neurokinin B immunoreactive neurons in the infundibular nucleus of aged men and women.

The secretory output of gonadotropin-releasing hormone (GnRH) neurons is critically influenced by peptidergic neurons synthesizing kisspeptins (KP) and neurokinin B (NKB) in the hypothalamic infundibular nucleus (Inf). These cells mediate negative feedback effects of sex steroids on the reproductive axis. While negative feedback is lost in postmenopausal women, it is partly preserved by the sustained testosterone secretion in aged men. We hypothesized that the different reproductive physiology of aged men and women is reflected in morphological differences of KP and NKB neurons. This sexual dimorphism was studied with immunohistochemistry in hypothalamic sections of aged human male (≥50 years) and female (>55 years) subjects. KP and NKB cell bodies of the Inf were larger in females. The number of KP cell bodies, the density of KP fibers, and the incidence of their contacts on GnRH neurons were much higher in aged women compared with men. The number of NKB cell bodies was only slightly higher in women and there was no sexual dimorphism in the regional density of NKB fibers and the incidence of their appositions onto GnRH cells. The incidences of NKB cell bodies, fibers, and appositions onto GnRH neurons exceeded several-fold those of KP-IR elements in men. More NKB than KP inputs to GnRH cells were also present in women. Immunofluorescent studies identified only partial overlap between KP and NKB axons. KP and NKB were colocalized in higher percentages of afferents to GnRH neurons in women compared with men. Most of these sex differences might be explained with the lack of estrogen negative feedback in aged women, whereas testosterone can continue to suppress KP, and to a lesser extent, NKB synthesis in men. Overall, sex differences in reproductive physiology of aged humans were reflected in the dramatic sexual dimorphism of the KP system, with significantly higher incidences of KP-IR neurons, fibers and inputs to GnRH neurons in aged females vs. males.

Short-term effect of fine particulate matter (PM 2.5) on daily mortality due to diseases of the circulatory system in Madrid (Spain).

INTRODUCTION: Owing to their small size, fine particles, i.e., those having a diameter ≤ 2.5 µm (PM(2.5)), have a high alveolar penetration capacity, thereby triggering a local inflammatory process with circulatory repercussion. Despite being linked to respiratory and cardiovascular morbidities, there is limited evidence of an association between this type of particulate matter and short-term increases in mortality. OBJECTIVE: The aim of this study was to analyse and quantify the short-term impact of PM(2.5) on daily mortality due to diseases of the circulatory system, registered in Madrid from 1 January 2003 to 31 December 2005. METHODS: An ecological longitudinal time-series study was conducted, with risks being quantified by means of Poisson regression models. As a dependent variable, we took daily mortality registered in Madrid from 1 January 2003 to 31 December 2005, attributed to all diseases of the circulatory system as classified under heads I00-I99 of the International Classification of Diseases-10th revision (ICD-10) and broken down as follows: I21, acute myocardial infarction (AMI); I20, I22-I25, other ischemic heart diseases; and I60-I69, cerebrovascular diseases. The independent variable was daily mean PM(2.5) concentration. The other variables controlled for were: chemical pollution (PM(10), O(3), SO(2), NO(2) and NO(x)); acoustic and biotic pollution; influenza; minimum and maximum temperatures; seasonalities; trend; and autocorrelation of the series. RESULTS: A linear relationship was observed between PM(2.5) levels and mortality due to diseases of the circulatory system. For every increase of 10 µg/m(3) in daily mean PM(2.5) concentration, the relative risks (RR) were as follows: for overall circulatory mortality, associations were established at lags 2 and 6, with RR of 1.022 (1.005-1.039) and 1.025 (1.007-1.043) respectively; and for AMI mortality, there was an association at lag 6, with an RR of 1.066 (1.032-1.100). The corresponding attributable risks percent (AR%) were 2.16%, 2.47% and 6.21% respectively. No statistically significant association was found with other ischemic heart diseases or with cerebrovascular diseases. CONCLUSION: PM(2.5) concentrations are an important risk factor for daily circulatory-cause mortality in Madrid. From a public health point of view, the planning and implementation of specific measures targeted at reducing these levels constitute a pressing need.

High dose-rate afterloading 192Iridium prostate brachytherapy: feasibility report.

BACKGROUND: RESULTS from localized prostate cancer series using seed implants have been most encouraging. However, with current techniques, inadequate dosimetry sometimes occurs. Remote afterloading high dose rate 192Iridium brachytherapy (HDR-Ir192) theoretically remedies some potential inadequacies of seed implantation by performing the dosimetry after the needles are in place. This study was undertaken to determine the feasibility of incorporating multifractionated HDR-Ir192 in the brachytherapy management of prostate carcinoma. METHODS: From October 1989 to August 1995, 104 patients were treated with a combination of multifractionated HDR-Ir192 and external beam. Patients ranged in age from 48-78 years, with a mean of 68.6 years. By TNM clinical stage, there were 1 T1b, 31 T1c, 28 T2a, 24 T2b, 9 T2c, 8 T3a, and 3 T3c lesions. For the group, the mean initial pretreatment PSA was 12.9 ng/ml (median 8.1), with 90% of the patients having had a pretreatment PSA greater than a normal value of 4.0 ng/ml. Patients with prostate volumes up to 105 cc were implanted. Treatment was initiated with perineal needle placement using ultrasound guidance. A postoperative CT scan was obtained to provide the basis for treatment planning. Four HDR-Ir192 treatments were given over a 40-h period, with a minimal peripheral dose (MPD) ranging from 3.00 to 4.00 Gy per fraction over the course of this study. Two weeks later, external beam radiation was added using 28 fractions of 1.80 Gy daily, to a dose of 50.40 Gy. RESULTS: Follow-up ranged from 10 to 89 months, with a mean of 46 months and median of 45 months. At various follow-up points, the patient numbers at risk were: 1 year, 101; 3 years, 69; 5 years, 28. The technique proved to be uniformly applicable to a wide range of prostate volumes and was very well tolerated by patients. Nearly all significant late in-field treatment complications were genitourinary in nature. Of the patients, 6.7% developed urethral strictures that were readily manageable. Changes in technique implemented in 1993 appear to have significantly lessened the incidence of this complication. Two patients developed significant uropathy within the first treatment year, but both resolved; 1 of these 2 patients had a prior TURP. Other bladder or rectal complications have been minimal. Using PSA progression as a marker of tumor response, approximately 84% of patients whose initial PSA was less than 20 ng/ml were free of progression at 5 years by actuarial analysis. CONCLUSIONS: We found the use of transperineal ultrasonography, postimplant CT-based dosimetry, coupled with adjustable dose delivery inherent to remote afterloading technology, to give unparalleled control in performing this complex brachytherapy task. Thus, it may be advantageous in certain clinical situations where the resultant MPD is needed to reliably cover the target volume, such as in patients with carcinomas at base locales, when the possibility of moderate to extensive intraprostatic tumor exists, and in patients with large glands. Early PSA data suggest that it may be effective as a definitive treatment with rates of adverse late tissue effects that are acceptable using current technique and doses described herein. Longer follow-up is needed to ascertain its position among the various treatment regimens for prostate carcinoma.

Extending the limits of lung cancer resection.

Patients with locally advanced bronchogenic carcinoma are often considered to have unresectable disease because of invasion into vital structures, or they undergo resection with questionable or involved margins, which results in local recurrence later. Brachytherapy (direct application of radioactive sources to the tumor bed) offers the potential to provide tumoricidal doses of radiation to the target area with minimal toxicity to surrounding structures. In this study, one of two different techniques of brachytherapy was utilized to treat 15 highly selected patients with histologically positive (n = 8) or suspicious (n = 7) margins after resection. The techniques were easy to apply and were not associated with any complications directly related to their use. One postoperative death resulted from a perforated peptic ulcer. In the remaining 14 patients, at a mean follow-up of 38 months, local control was complete in 12 (86%) patients, and 8 patients are alive, with 7 free of disease. Thoracic brachytherapy may offer the potential for cure to patients whose disease would otherwise be considered inoperable.

Malignant biliary obstruction: intracavitary treatment with a high-dose-rate remote afterloading device.

A new technique of intracavitary brachytherapy for malignant biliary obstruction is presented. The technique involves the use of a high-dose-rate remote afterloading device, which offers all the advantages of conventional brachytherapy with the added benefit that the dose can be delivered in a single treatment over a few minutes. The potential problems associated with conventional brachytherapy are thereby minimized.

Suppressor cells induced by total lymphoid irradiation affect proliferation and lymphokine production of murine T helper cell clones.

A key response to antigen is the activation of helper T cells to release lymphokines which stimulate and effect the immune reaction. This T cell population can secrete many different factors with diverse, often multifunctional roles, such as amplifying T or B cell antigen responses or being effectors of cell mediated delayed type hypersensitivity. Among these lymphokines are gamma-interferon (gamma-IFN), interleukin-2 (IL-2), or T cell growth factor, and lymphotoxin (LT) which has cytotoxic activity against a variety of cells. Immune suppression in mice following total lymphoid irradiation (TLI) has been correlated with the presence in lympho-reticular tissues of an antigen non-specific, null suppressor cell. This study examined what effects radiation induced suppressor cells had upon the in vitro activation and lymphokine responses of the ovalbumin (OVA) specific T helper cell clone, 153E6, following antigen presentation. Splenocytes from TLI treated mice obtained early in the post-irradiation period exerted a pan-inhibitory effect upon OVA induced 153E6 proliferation and its concomitant release of gamma-IFN, LT, and IL-2. As the interval from irradiation increased, splenocytes from TLI treated mice showed persistent suppression of 153E6 proliferation and gamma-IFN release, but had rapidly diminishing effects on the T cell's capacity to produce LT and IL-2. These findings suggest that suppressor cells induced by TLI have a marked inhibitory effect in vivo upon T helper cell proliferative responses to antigen and the production of various T helper cell lymphokines necessary to mediate the immune response. Such processes could contribute to the immunosuppressive effects of extensive nodal irradiation.

A clinical and histopathologic analysis of the results of conservation surgery and radiation therapy in stage I and II breast carcinoma.

One hundred eighty women with clinical Stage I or II operable breast carcinoma were treated by radiotherapy following local tumor excision at Yale-New Haven Hospital through 1980. With a median follow-up time of 6.9 years, the actuarial 5-year overall and disease-free survival rates were 82% and 78%, respectively. The 5-year actuarial breast-recurrence-free survival rate was 92%. Several clinical-histopathologic features and treatment parameters were assessed for their significance as predictors of local breast failure or distant relapse. Cox lifetable regression analysis showed that patients with clinical Stage II carcinomas had significantly worse overall and relapse-free survival rates, but clinical stage alone had no effect on the rate of breast recurrence. Furthermore, a decrease in overall and disease-free survival was evident when necrosis was present in the tumor or when patients had an infiltrating lobular carcinoma. Breast recurrence-free survival was also influenced adversely by the presence of these two tumor features, especially when either tumor necrosis or infiltrating lobular carcinoma was found in conjunction with clinical Stage II lesions. Other histologic features such as grade, vascular invasion, perineural invasion, or the presence of an intraductal component of carcinoma did not affect outcome, nor did the treatment techniques employed appear to have a differential effect.

Reactions of tumors and normal tissues in mice to irradiation in the presence and absence of a perfluorochemical emulsion.

The effect of pre-treatment with the perfluorochemical emulsion, Fluosol-DA, on the radiation response of normal tissues and EMT6 mammary tumors in BALB/c mice was examined. Pre-treating tumor-bearing mice with .015 ml/g of Fluosol and 30 min of carbogen (95% O2/5% CO2) increased the number of tumor cells killed by irradiation with doses of 2.5-20 Gy; the change in the radiation dose-response curve was consistent with a reduction in the hypoxic fraction. Fluosol did not alter the response of tumors in air-breathing or N2-asphyxiated mice and carbogen alone did not alter the radiation response of this tumor significantly. Carbogen treatments 5-60 min in duration produced similar enhancements of tumor radiosensitivity in Fluosol-treated animals. Pre-treatment with Fluosol plus carbogen also increased the number of tumor cells killed by a fractionated regimen of four 2.5 Gy fractions given over 2 days. Pre-treatment with Fluosol-DA plus carbogen, therefore, increased the antineoplastic effects of radiotherapy in both single-dose and multi-fraction radiation regimens. In contrast, Fluosol did not increase the effect of radiation on the partially committed (CFU-GM) or pluripotent (CFU-S) stem cells of the bone marrow or on the CFU-GM of the spleen. The radiation response of the skin was only slightly enhanced by pre-treatment with Fluosol plus carbogen. These data show that treatment of mice with perfluorochemical emulsions plus carbogen can produce therapeutic gain by improving the radiation response of solid tumors, without producing an equivalent increase in the radiation response of potentially dose-limiting normal tissues. These findings encourage further evaluations of these agents as adjuncts to clinical radiotherapy.

Iodine-125 implants for carcinoma of the prostate.

One hundred-thirteen patients underwent Iodine-125 prostate implant and lymphadenectomy at Yale-New Haven Hospital from 1974 through 1980. The distribution by clinical stage was: 7 Stage A2, 86 Stage B, and 20 Stage C patients. Ninety-four patients had a negative lymphadenectomy (N-) and 19 patients (17%) had metastatic disease in the pelvic lymph nodes (N+). The actuarial 5-year survival for all 113 patients was 87% (+/- 6%: 95% confidence limits). Sixty-five percent of our 113 patients are disease free (NED) from 2 to 9 years following implant. Sixty-seven (N-) patients with clinical Stage B disease, whose tumors were either well differentiated or moderately well differentiated, have an actuarial 5-year NED survival of 84% (+/- 8%). Twenty (N-) patients with either clinical Stage C disease or poorly differentiated tumors have an actuarial 5-year NED survival of only 31% (+/- 20%). For the 19 (N+) patients, the actuarial 5-year NED survival is 38% (+/- 22%). Local tumor control was 85% for all Stage B patients and 75% for all Stage C patients. Only 10 patients (9%) have developed long-term gastrointestinal or genitourinary complications following their implant. Iodine-125 implant appears to be a reasonable alternate form of therapy in highly selected groups of patients with carcinoma of the prostate.

Hodgkin's disease manifesting as Hashimoto's thyroiditis.

Hodgkin's disease can be manifested in ways other than lymphadenopathy. Two patients had symptoms related to thyroid enlargement and were initially believed to have Hashimoto's thyroiditis on the basis of markedly elevated thyroid antibody titers. Involvement of the thyroid region by Hodgkin's disease was eventually diagnosed.

Aryl-hydrocarbon hydroxylase activity in lymphocytes from lung cancer patients and normal controls.

A radiometric assay for the determination of AHH activity in lymphocytes is described. Subjects included eleven male patients with histologically verified lung cancer (nine squamous cell carcinoma and two adenocarcinoma) and eleven age- and sex-matched controls. Lung cancer patients exhibited considerably greater variation and elevated levels of both AHH activity per se and AHH activity adjusted for total cellular DNA than control individuals. Methodology for AHH determinations and implications for lung cancer epidemiology and control are discussed.