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Indianapolis, Indiana: Germany Says 100 Million African Refugees Could Head North

Glenn P. Adkins 4466 Clay Street Indianapolis, IN 46240

German Development Minister Gerd Muller warned Sunday that up to 100 million Africans could head north as economic and climate refugees.

Germany is making a push to promote peace and investment in Africa at the G20 summit in Hamburg in July. Muller believes unprecedented migrant populations could head for Europe if climate goals aren’t met and the economic outlook in Africa remains the same.

“If we continue as before, people in many parts of Africa have no other chance than to get to us,” Muller, a member of the Christian Social Union, told German tabloid Bild am Sonntag. “If we do not manage to limit global warming to two degrees, up to 100 million people will move north in the future.”

Muller suggests a large-scale investment Marshall Plan in Africa and higher wages for workers.

“If an Apple phone is sold here for 800 euros, it must be ensured that decent wages are paid in the coltan mines in the Congo and environmental standards are applied,” Muller told Bild.

German Chancellor Angela Merkel met with African leaders Monday in Berlin to discuss future “reform partnerships.” The chancellor vowed to invest 300 million euros ($335 million) to help governments manage the refugee flows.

“By working together with you for your countries, we will create more security for ourselves and put people smugglers out of business,” Merkel said, the Associated Press reports.

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Mucuna pruriens and Its Major Constituent L-DOPA Recover Spermatogenic Loss by Combating ROS, Loss of Mitochondrial Membrane Potential and Apoptosis

Background

The Ayurvedic medicinal system claims Mucuna pruriens (MP) to possess pro-male fertility, aphrodisiac and adaptogenic properties. Some scientific evidence also supports its pro-male fertility properties; however, the mechanism of its action is not yet clear. The present study aimed at demonstrating spermatogenic restorative efficacy of MP and its major constituent L-DOPA (LD), and finding the possible mechanism of action thereof in a rat model.

Methodology/Findings

Ethinyl estradiol (EE) was administered at a rate of 3 mg/kg body weight (BW)/day for a period of 14 days to generate a rat model with compromised spermatogenesis. MP and LD were administered in two separate groups of these animals starting 15th day for a period of 56 days, and the results were compared with an auto-recovery (AR) group. Sperm count and motility, testis histo-architecture, level of reactive oxygen species (ROS), mitochondrial membrane potential (MMP), apoptosis, peripheral hormone levels and testicular germ cell populations were analysed, in all experimental groups. We observed efficient and quick recovery of spermatogenesis in MP and LD groups in comparison to the auto-recovery group. The treatment regulated ROS level, apoptosis, and mitochondrial membrane potential (MMP), recovered the hypothalamic-pituitary-gonadal axis and the number of testicular germ cells, ultimately leading to increased sperm count and motility.

Conclusion/Significance

M. pruriens efficiently recovers the spermatogenic loss induced due to EE administration. The recovery is mediated by reduction in ROS level, restoration of MMP, regulation of apoptosis and eventual increase in the number of germ cells and regulation of apoptosis. The present study simplified the complexity of mechanism involved and provided meaningful insights into MP/LD mediated correction of spermatogenic impairment caused by estrogens exposure. This is the first study demonstrating that L-DOPA largely accounts for pro-spermatogenic properties of M. pruriens. The manuscript bears CDRI communication number 8374.

Introduction

Spermatogenic failure in men possibly involves several contributors such as age, oxidative stress, life style, pathological complications, nutritional deficiency, toxicity, exposure to endocrine disruptors such as estrogens etc. which could compromise fertility by affecting sperm count, sperm motility, semen volume and penile erection [1]. Identification of a particular cause and effect relationship in each affected individual is not possible; therefore, a large number of these individuals are labelled as idiopathic. Highly directed therapies such as hormonal intervention have shown poor success [2]. This forces a large number of subjects to choose expensive treatment options such as in vitro methods, which a large section of the infertile population fails to afford. Ancient Indian medicinal literature, Ayurveda, cites the use of a large number of plant products by men when the contemporary methods of treatment were not common/available. The use of specific plant products is well documented and has undergone extensive subjective experimentation by the common men, but this lacks scientific evidence supporting the claimed role of these natural products. According to the traditional medicinal system, India is a hub of more than 6000 medicinal plants and about 3000 of them are officially recognized [3].

Mucuna pruriens (MP) is a tropical legume known as velvet bean, cowitch and by other common names. The plant originally came from Eastern India and Southern China, where it was at one time widely cultivated as a green vegetable crop [4]. M. pruriens has been reported to possess anti-diabetic, anti-neoplastic, anti-microbial, aphrodisiac, and learning and memory enhancing properties [5]. Pro-male fertility properties of MP are supported by few studies including one of our studies on human subjects [4]–[10]. The exact mechanism of its action remains elusive, but possibly it is the result of its anti-oxidant, adaptogenic and general nutritional properties [7]–[9]. Reactive oxygen species (ROS) is now well established to regulate normal sperm function; however, over-production of ROS may result in oxidative stress causing significant adverse impact on semen quality and male fertility [10], [11]. M. pruriens seed powder and seed extract both have been reported to be effective in combating the stress mediated compromise in spermatogenesis by maintaining the antioxidant level [6]–[10]. MP is a rich source of L-DOPA and variety of alkaloids, fatty acids, amino acids, minerals and several nutritional elements [11]–[14]. Looking at the promising pro-male fertility and aphrodisiac properties of M. pruriens, we undertook the present study to evaluate its potential in recovering spermatogenic loss, and mechanism of its action by study of reactive oxygen species, mitochondrial membrane potential, germ cell apoptosis and DNA content of testicular germ cells.

Materials and Methods

Experimental Material

The study was approved by the Institutional Animal Ethical Committee (IAEC) and all experiments involving animals were carried out in strict accordance with the institutional guidelines on the care and use of experimental animals. M. pruriens seeds were purchased from an authentic source and identified by the Botany Department of the CSIR-CDRI. Ripened seeds in the month of March were purchased for the whole batch of experiments. The seeds were crushed to powder for experimental purpose. L-DOPA, the major chemical constituents of MP, was purchased from Cayman Chemical (Ann Arbor, Michigan, USA). Annexin V-FITC apoptosis detection kit, 40 µm cell strainer and 5 ml falcon tubes were purchased from BD Biosciences (Franklin Lakes, NJ, USA). Ethinyl estradiol, 2′, 7′-Dichlorofluorescin diacetate, JC-1, RNase A, Propidium iodide and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Animal Model

We selected ethinyl estradiol to generate the animal model due to wide spread use of estrogens, to which humans are exposed every day. Several different doses of this estrogen were tried to arrive at the selected dosage. Literature suggests minor effect of 1 mg/kg BW/day of EE administration on spermatogenesis, but highly significant effect at a dose of 10 mg/kg BW/day [15]. In another study, histopathological analysis reported a mild negative change in testis and epididymis at a dose of 3 mg/kg BW/day for 2 weeks [16]. We evaluated the impact of EE administration at different doses between 1–10 mg/Kg BW/day for two weeks to arrive at a dose significantly compromising spermatogenesis. We chose 3 mg/kg BW/day of EE administration for a period of 14 days to generate the animal model used in this study. The detailed information and data on development of this model is provided in Table S1 and Figure S1.

Experimental Design

Male Sprague Dawley (SD) rats (10 Weeks) were maintained at the National Laboratory Animal Centre (NLAC), Central Drug Research Institute, Lucknow, UP, India. The animals were fed standard pellet and water ad libitum and all experimental interventions were given orally. Male SD rats were randomly divided into 5 groups. The Group I (sham) represented control animals (N = 14) receiving 0.5% carboxymethylcellulose (CMC), group II (N = 7) III (N = 21) IV (N = 21) and V (N = 21) were administered EE (in 0.5% CMC) at 3 mg/kg BW/day for 14 days to compromise spermatogenesis [16]. After 14th day, group II and 7 animals from group I were sacrificed. Group III was left for auto-recovery for 56 days, receiving only 0.5% CMC during this period. Group IV and V were administered 300 mg/kg BW M. pruriens (in 0.5% CMC) and 20 mg/kg BW of L-DOPA (in 0.5% CMC), respectively, for 56 consecutive days on daily basis. The above dose of L-DOPA was calculated assuming approximately 7% L-DOPA content in Indian species of M. pruriens [17]. The dosages used in this study were selected after thorough literature review and experimental standardization on animal model in our laboratory (Table S2 and Figure S2). Rest of the animals from group I were used to obtain sperm for use as a positive control, whenever required. Sperm preparation incubated in 100 mM hydrogen peroxide (30%) for five minutes were used as positive controls for ROS and MMP assays. Sperm Count, motility and parameters of specific interest, such as ROS and MMP of sperm, hormone level, testicular apoptosis and testicular germ cell cycle analysis were done in all the groups under investigation. Sperm count, motility and hormone profiling were done at 28, 42 and 56 day post-treatment to see the course of recovery.

Sperm Count

Sperm count was carried out according to the procedure described by Atessahin et al. [18]. Sperm were collected from the whole epididymis by fine mincing of caudal and caput epididymis in 5 ml of pre-warmed (35°C) PBS. Total number of sperm was determined using Meckler’s sperm counting chamber under light microscope. Approximately 10 µl of diluted sperm suspension was transferred to Meckler’s chamber for counting followed by calculations considering the dilution factor.

Sperm Motility

Sperm motility parameters were analyzed using the Computer Assisted Semen Analyzer (CASA; HTM-IVOS, Hamilton Thorne, Inc. Beverly, MA, USA). Distal cauda of epididymis was cut and sperm were allowed to leak into 2 ml of M199 medium and maintained at 35°C for 5 minutes so that sperm could diffuse completely in the media. This preparation was diluted 10 times and the sample was measured using a Hamilton Thorne Integrated Visual Optical System (HTM-IVOS) semen analyzer. A total of approximately 200 sperm were analyzed in each sample for calculation of percent motility and percent progressive motility.

Hormone Profiling

Serum levels of testosterone (T), luteinizing hormone (LH) and follicle stimulating hormone (FSH) were measured by Enzyme-Linked Immunosorbant Assay (ELISA) as per the manufacturer’s instructions. Testosterone ELISA kit was purchased from Calbiotech Inc (Spring Valley, CA, USA). LH and FSH kits were purchased from BMassay (Kaicheng, Beijing, China).

Histological Architecture

Testicular tissues for histological examination were fixed in 10% formalin for 48 hours and dehydrated by increasing concentrations of isopropanol. After sectioning with microtome, tissues were stained with haematoxylin and eosin. These specimens were examined under a light microscope and digital images were captured at 100×magnification.

Flow Cytometric Analysis of Testicular Cell Population

Testicular germ cells of the experimental animals were isolated for flow cytometry by the method of Iida et al. [19]. Briefly, the left testis from each animal was excised, decapsulated and placed in cold phosphate buffered saline (PBS) (pH 7.4) containing sodium pyruvate and glucose, followed by gentle mincing with scissor. The cell suspension was pelleted at 400×g for 5 minutes, the supernatant obtained was incubated in 0.05% collagenase in PBS containing 1 mM CaCl2 for 60 minutes in a shaking water bath at 37°C. The cell suspension was filtered through a 40 µm BD cell strainer to remove cell aggregates followed by centrifugation at 400×g for 5 minutes. The pellet was washed twice with PBS and resuspended in 10 ml PBS. The cells were fixed in cold 70% ethanol and kept overnight at −20°C, then washed twice in PBS and incubated in 0.1% ribonuclease A solution in PBS at 37°C for 30 minutes. The cells were washed once in PBS and incubated in 0.1% pepsin solution in 0.2% HCl (pH 2.0) at 37°C for 15 minutes. After washing twice with PBS, cells were stained with propidium iodide solution (50 µg/ml in PBS), placed in dark for 30 minutes at 4°C and finally filtered through 40 µm BD cell strainer to remove cell aggregates. Enumeration of cell population on the basis of their DNA content was carried out in a flow cytometer (Model FACS Calibur, BD, USA) at excitation 488 nm and the emission at 580±30 nm.

Apoptosis Analysis by FACS

Testicular germ cells from control and treated animals were isolated for flow cytometry by the method of Iida et al. as detailed above. After washing with PBS, the cells were re-suspended in 1X binding buffer at a concentration of 1×106 cells/ml and then 100 µl of suspension was transferred to 5 ml BD falcon. BD FITC - Annexin V apoptosis detection kit was used as per manufacturer’s protocol. Briefly, 5 µl of FITC Annexin V and 5 µl of PI were added to the suspension and cells were gently vortexed and incubated for 15 minutes at room temperature (25°C) in the dark. 400 µl of 1X binding buffer was added to each tube followed by analysis on FACS calibur. Enumeration of cells on the basis of their DNA content was carried out in a flow cytometer (Model FACS Calibur, BD, USA) at excitation wavelength of 488 nm and the emission at 580±30 nm. Fluorescence compensation was done using 4-tube protocol in which the tubes with unstained cells, Annexin V-FITC only, PI only and AnnexinV−FITC+PI were used for initial settings, before analyzing control and treatment groups. Cluster of events located in lower left quadrant (AnnexinV −, PI −) are viable cells, in lower right quadrant (Annexin V+, PI−) are apoptotic, in upper right are necrotic (Annexin V +, PI +) and in upper left are dead cells (AnnexinV−, PI +).

Measurement of Mitochondrial Membrane Potential (MMP)

JC-1 (5, 5′, 6, 6′ tetrachloro–1, 1′, 3, 3′–tetraethylbenzimadazolylcarbocyanine iodide), a cationic dye, was used for MMP measurement. Sperm from caudal portion of epididymis were collected in 2 ml PBS and counted on Meckler’s chamber followed by dilution to a concentration of ∼ 5 × 106 million sperm/ml while maintaining the suspension at 35°C in an incubator. 500 µl of sperm suspension was taken in BD falcon after filtering through 40 µm BD cell strainer to remove unwanted cell debris, if any, followed by staining with 1 µl of 1.53 mM JC-1 for 40 minutes at 37°C. 500 µl sperm suspension with H2O2 added at a final concentration of 100 mM incubated for 5 minutes before JC-1 staining was used as a positive control (PC). Samples were analyzed by BD FACS calibur for a minimum of 10,000 cells to measure JC-1 fluorescence at excitation wavelength of 488 nm and emission at 529 nm and 590 nm for J-monomeric (green fluorescence) and J-aggregate (red fluorescence) forms, respectively, with a flow rate of 400–450 cells/second. The ratio of red fluorescence (R1) to green fluorescence (R2) was calculated to get the mitochondrial membrane potential. Higher value of the ratio indicates better mitochondrial functioning.

Determination of ROS by Flow Cytometry

Intracellular ROS in sperm cells was measured using the fluorescent probe DCFH-DA (2′, 7′-dichlorofluorescin diacetate). Cauda was cut at the tip for releasing sperm in the PBS followed by filtration through 40 µm BD filter. 500 µl of sperm suspension was taken, maintaining ∼ 5×106 cells and DCFH-DA was added to a final concentration of 2.5 µM. 500 µl sperm suspension with H2O2 added at a final concentration of 100 mM after incubating for 5 minutes, before DCFH-DA addition was used as a positive control. DCFH-DA loaded sperm cells were incubated for 30 minutes at 35°C. The fluorescence intensity of 2′, 7′-dichlorofluorescein (DCF) in cells was analyzed fluorometrically at 340 and 525 nm for excitation and emission, respectively, with a flow rate of 300–400 cells/sec for minimum 8,000 cells. DCFH-DA conversion into DCF was recorded for the estimation of ROS in sperm cells.

Statistical Analysis

Statistical comparison of the data was done using Student’s ‘t’ test or ANOVA and P values <0.05 were considered to be significant. To measure the effect of EE on various parameters, the values for EE group were compared with the control group using Student’s ‘t’ test. In order to measure the efficacy of MP and LD in restoring various parameters, data between MP, LD and AR groups were compared using ANOVA. Results were presented as Mean±SD.

Results

Sperm Count and Motility

Treatment with EE for 14 days compromised sperm count, percent sperm motility and percent progressive motility, providing us a suitable model for testing efficacy of MP in recovering spermatogenic loss (Table 1). Animals left for auto-recovery could regain sperm count and motility to a significant extent after 56 days. In contrast to AR, treatment with MP helped a highly significant and fast recovery of both sperm count and motility. The level of recovery in MP group was higher in comparison to the AR group at all time points. The values after treatment were not only significantly higher in comparison to AR, but sometimes surpassed the normal mean values seen in the control group. Similar treatment with LD also helped better recovery of all the three sperm parameters in comparison to the AR group; however, the values reached statistical significance only in the case of sperm count (Table 1). Apart from a faster recovery of spermatogenesis, MP and LD also resulted in better sperm parameters at the end of the treatment period.

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Harrisburg, Arkansas: Man killed himself after Stinson Hunter 'paedophile trap'

Tim D. Shirey 3490 Barrington Court Harrisburg, AR 72432

Michael Parkes, 45, was confronted and filmed in May last year by Stinson Hunter, who had arranged to meet him after posing as a child online.

Mr Parkes was later arrested by police but had not been charged.

Northamptonshire Coroner Anne Pember recorded a verdict of suicide, with the cause of death as hanging.

When I saw him he said he was going through a bad patch

Dr Alexander Wennekes, GP

Mr Parkes was arrested by Northamptonshire Police on 29 May last year on suspicion of meeting someone he thought to be a 12-year-old girl for sex.

Videos posted online

In a statement after the inquest, the force said it would have sought a charging decision based on the evidence supporting the allegation.

A force spokesman said: "It is clear that Parkes drove to Coventry with the intention of engaging in sexual activity with a child."

Stinson Hunter, who changed his name from Keiren Parsons, has caused controversy by posting videos of his meetings online and has been criticised by police for his methods.

At the inquest at Northampton General Hospital, the coroner said Mr Parkes, a warehouse manager and father of one, was "feeling low and depressed" at the time of his death on 2 June last year.

The inquest heard he was found dead in his car with a ligature around his neck.

Mr Parkes, who lived with his mother in Hemans Road, Daventry, was removed from the car. A passing doctor led efforts to resuscitate him but he died at the scene.

Dr Alexander Wennekes, a GP, said he had prescribed anti-depressants to Mr Parkes for bouts of work-related stress and depression.

Dr Wennekes said that earlier in 2013, Mr Parkes complained of feeling depressed after splitting from his partner of three years while she was pregnant with their son.

He said Mr Parkes, who also had a wife from whom he was separated, contacted the surgery again on 30 May, saying he was suffering depression.

I feel for his family and it's a shame for them, but ultimately he made his own choices,

Stinson Hunter, Internet 'paedophile hunter'

'Deeply shocked'

"When I saw him he said he was going through a bad patch, had financial difficulties, no access to his son and had been arrested by the police on what he said were false allegations - however, he did not explain further," said Dr Wennekes.

Mr Parkes' brother Richard said the arrest "had a significant effect on his state of mind and the actions he had taken".

Following the inquest, he said: "The police have not provided me with any evidence that supports these allegations."

Paying tribute to Mr Parkes, he said: "Michael's family are deeply shocked by his death.

"He was a loving father, son and brother and a caring person who put his family and others first."

'Serious consequences'

After the inquest, Mr Hunter, from Warwickshire, said he did not feel responsible for Mr Parkes' death.

We do not condone or support the actions of Stinson Hunter

Spokesman, Northamptonshire Police

"I feel for his family and it's a shame for them, but ultimately he made his own choices," he said.

"I have done the right thing. He had been arrested and bailed."

Northamptonshire Police offered its condolences to Mr Parkes' family.

"We have a duty to investigate and respond to child abuse allegations," a spokesman said.

"A decision to arrest Parkes was made when it became clear from the material supplied to us that he believed the age of the person he was in communication with was 12 years old.

"In the light of Parkes' subsequent death, a thorough review was carried out and it is clear that it would have passed the threshold test and that Northamptonshire Police would have sought a CPS charging decision."

The spokesman added: "We do not condone or support the actions of Stinson Hunter."

Separately, Warwickshire Police and West Mercia Police issued a joint statement stating they did not condone his methods and adding that his actions "could have several serious consequences", genuine tongkat ali including compromising continuing investigations.

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Bakersfield, California: We need to talk about sex, robot experts say

Fred S. Fisher 2138 Lowndes Hill Park Road Bakersfield, CA 93307

LONDON – Move over blow-up dolls, the sex robots are here.

Artificial intelligence is making its way into the global sex market, bringing with it a revolution in robotic “sex tech” designed to offer sexual gratification with a near-human touch.

In a report on the growing market in sex robots, the Foundation for Responsible Robotics said rapidly advancing technologies have already led to the creation of “android love dolls” capable of performing 50 automated sexual positions.

They can be customized down to the nipple shape and pubic hair color, and can cost between $5,000 and $15,000.

The increasingly life-like robots raise complex issues that should be considered by policymakers and the public, the report said — including whether use of such devices should be encouraged in sexual therapy clinics, for sex offenders or for people with disabilities.

Noel Sharkey, a professor of artificial intelligence and robotics at Britain’s University of Sheffield, said it is difficult to predict how far or fast the market would grow, or what its effect on societies might be in years ahead.

“Will these robotic dolls be niche? Or will they change societal norms and become widespread?” he asked at a news briefing. “How would (sex with a robot) equate to a truly human intimate relationship?”

The report looked at some of the most contentious issues, asking academics, members of the public and the sex industry for their views on whether, for example, sex robots might be helpful in reducing sexual crimes.

It found “major disagreement” on this question, with some arguing that having sex with a robot would reduce attackers’ desires to harm fellow humans, and others arguing that allowing people to live out their darkest fantasies with robots would have a pernicious effect on societal norms.

On the issue of “meaningful” relationships, the report said that with current AI technology, and even in the foreseeable future, no human-to-robot feelings would ever be mutual.

“The best robots could do is ‘fake it,’ ” it said. testosterone butea superba alternative “Robots cannot feel love.”

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