Maternal mortality ratio in low- to middle-income countries (LMIC) is 14 times higher than in high-income countries. This is partially due to lack of antenatal care, unmet needs for family planning and education, as well as low rates of birth managed by skilled attendants. While direct causes of maternal death such as complications of hypertension, obstetric haemorrhage and sepsis remain the largest cause of maternal death in LMICs, cardiovascular disease emerges as an important contributor to maternal mortality in both developing countries and the developed world, hampering the achievement of the millennium development goal 5, which aimed at reducing by three-quarters the maternal mortality ratio until the end of 2015.Systematic search for cardiac disease is usually not performed during pregnancy in LMICs despite hypertensive disease, rheumatic heart disease and cardiomyopathies being recognised as major health problems in these settings. New concern has been rising due to both the HIV/AIDS epidemic and the introduction of highly active antiretroviral therapy. Undetected or untreated congenital heart defects, undiagnosed pulmonary hypertension, uncontrolled heart failure and complications of sickle cell disease may also be important challenges. This article discusses issues related to the role of cardiovascular disease in determining a substantial portion of maternal morbidity and mortality. It also presents an algorhitm to be used for suspected and previously known cardiac disease in pregnancy in the context of LIMCs. The 2010 Millennium Development Goals summit concluded with an action plan to accelerate progress on maternal and child health. The target of the millennium development goal 5 was to reduce by three-quarters the maternal mortality ratio and achieve universal access to reproductive health between 1990 and the end of 2015. A 2013 report by the United Nations states: ‘The maternal mortality ratio dropped by 45 per cent between 1990 and 2013, from 380 to 210 deaths per 100 000 live births. All regions have made progress but accelerated interventions are required in order to meet target.’1The maternal mortality ratio in low- to middle-income countries (LMIC) is 14 times higher than in high-income countries (HIC).HIC such as the United States of America and the United Kingdom have reported decreases in direct causes of maternal mortality, but deaths due to cardiovascular disease have remained unchanged or are increasing.2) and seven had an enlarged heart (the median heart weight at autopsy was 390 g).SADS is defined as sudden death in an adult where no cause is identified. It is believed that obesity, cardiac hypertrophy and severe atherosclerosis can cause arrhythmia and sudden death.PPCM is a potentially life-threatening heart disease emerging towards the end of pregnancy or in the first months postpartum in previously healthy women.Treatment recommendations rely on standard acute heart-failure therapy. After the acute phase, in addition to the standard treatment of chronic heart failure, novel disease-specific strategies, such as bromocriptine, should be considered. PPCM in itself is a prothrombotic condition and embolic events leading to strokes are common. Therefore, all patients with an ejection fraction (EF) < 35%, those receiving bromocriptine, and particularly if a thrombus has been visualised on echocardiography, should be anticoagulated with intravenous heparin or low-molecular weight heparin antepartum, and receive warfarin after delivery.The incidence of fatal ischaemic heart disease (IHD) in pregnancy ranges between 0.48 and 0.76 per 100 000 pregnancies. The most common presenting symptom in pregnancy is chest pain, which is present in 95% of women with IHD.Coronary artery dissection and thromboembolic coronary events are the most common causes of IHD reported in pregnancy.In the same United Kingdom registry for the 2006–2008 triennium there were seven maternal deaths due to aortic dissection. In most cases, patients presented with severe chest or interscapular pain requiring opiate analgesia, and the diagnosis was delayed as appropriate investigations were not performed.Hormonal changes and increased haemodynamic stress predisposes to aortic dissection in pregnancy, but the exact mechanism is unclear.Heart disease is a common problem in pregnancy in LMICs;Risk factors such as hypertension, obesity and diabetes are increasingly important, occuring in high numbers in some countries, urban settings and specific sub-populations in sub-Saharan Africa, Asia and Latin America. In Africa, hypertension is most frequently observed in both rural and urban communities, with prevalence rates in young populations ranging from 9.3 to 48.1%.In LMICs, a search for cardiac disease in pregnant women is not performed routinely although hypertensive disease, rheumatic heart disease and cardiomyopathies are recognised as the largest drivers of maternal mortality. Among the cardiomyopathies, peripartum, Chagas disease and endomyocardial fibrosis present specific challenges due to their poor prognosis and high prevalence in some geographical areas. Cardiovascular manifestations of HIV/AIDS is a major concern due to the higher prevalence of infection in African women and lower mean age of those affected, compared to men.17Undetected or untreated congenital heart defects continue to be diagnosed during pregnancy, which is witness to the fact that advances in paediatric cardiology and cardiac surgery over the last decades have benefited disproportionately women from developed countries, compared to those living in the developing world. In fact, pulmonary hypertension and severe heart failure complicating pregnancy are a common presentation when surviving girls reach their reproductive years. In specific endemic 3regions, sickle cell disease and other haemoglobinopathies may constitute important challenges by increasing the risk of thromboembolic complications due to the hypercoagulability caused by enhanced platelet function, activation of the coagulation cascade and impaired fibrinolysis in pregnant women.18n = 8), with pregnant women with congenital heart disease experiencing higher mortality rates.22Data on mortality rates of pregnant women with cardiac disease vary widely among LMICs, as data from hospitalbased studies with variable designs and different geographical contexts suggest. Hypertension (pregnancy in hypertensive women and hypertension aggravated by pregnancy) is probably the single most important cardiovascular risk factor linked to adverse maternal and neonatal outcomes in LMICs.Although data from Africa are scarce, maternal morbidity and mortality related to heart disease may reach unacceptably high rates. A systematic review of pre-existing cardiac disease inpregnant women in South Africa found seven studies where the prevalence of heart disease ranged from 123 to 943 per 100 000 deliveries, with a median prevalence of 616 per 100 000.25et al. studied 225 women presenting to a single tertiary care centre in South Africa with cardiovascular disease in pregnancy or within six months’ postpartum, showing that 54% of pregnant women presented to specialised care for the first time with a gestational age over 24 weeks.n = 7) and prosthetic valve complications (n = 2). Interestingly, eight out of nine deaths occurred outside the 42-day maternal mortality report period, meaning that they were not considered in the statistics as maternal deaths.Sliwa In LMICs, rheumatic heart disease contributes to 30% of the cardiovascular disease seen in pregnancy and remains an important determinant of morbidity and mortality.30p < 0.01). Among 1 825 women of childbearing age (12–51 years), only 65 (3.6%) were on contraception, reflecting the poor provision of family planning and pre-pregnancy advice for women with heart disease that occurs in many regions of the world.32The participating countries were grouped into three income categories according to 2011 World Bank definitions: low-income countries , lower-middle-income countries , and uppermiddle- income countries (Namibia and South Africa). There was no difference in the predominance of females in the three groups: 728/1 110 (65.8%) 867/1 370 (63%) and 616/863 (71.3%), respectively. However, a statistically significant difference was found in the proportion of women in child-bearing years between the three groups of countries  all pregnant women should be screened at booking for underlying medical or surgical conditions; (2) women with known or recently detected cardiovascular disease should undergo risk assessement based on an algorithm Socio-economic and demographic factors such as persistently high fertility rates, social pressure to conceive, insufficient access to contraceptive methods, as well as social or familial ostracism towards women who use contraception, may further contribute to increasing the risk of death due to cardiac disease, even in women who have been diagnosed.34Available data on maternal mortality rates reveal the pre-imminence of cardiovascular disease as the most important medical cause of non-obstetric maternal death in both developed and developing countries. Failure to systematically search for cardiac disease in pregnant women has led to late diagnosis and high rates of fatal complications. Therefore active screening for cardiac disease in pregnant women is warranted, if the millennium development goal of reducing the maternal mortality ratio is to be achieved.In LMICs algorithms for cardiac screening of pregnant women should consider the unique profile of cardiovascular disease, including rheumatic heart disease, cardiomyopathies, HIV/AIDS, haemoglobinopathies and undetected/untreated congenital heart defects. Such active strategies for suspected and previously known cardiac disease in pregnancy are expected to prevent a substantial proportion of maternal morbidity and mortality.


Long non-coding RNAs (lncRNAs) have emerged as important regulators of gene expression in a variety of biological process and in multiple species. In plants, they are transcribed by different RNA polymerases and show diverse structural features. With the aid of next-generation sequencing technologies, a large number of lncRNA have been identified in model plants as well as in crops. This review focuses on the demonstration that lncRNAs control root system architecture, notably in response to phosphate availability, through regulation of transcription, alternative splicing, microRNA activity, messenger RNA stability and translation, illustrating remarkable diversity in their roles in regulating developmental plasticity. COOLAIR and COLDAIR (FLOWERING LOCUS C (FLC). Sequencing-by-synthesis technologies has revealed lncRNAs as a significant component of the transcriptome in a number of plant species , requiring phosphate inputs as fertilizer or else limiting agricultural production. Low Pi availability in the soil results in Pi deficiency that triggers a cascade of developmental responses, which reprogram the root system architecture. This developmental plasticity allows roots to favor exploration of the shallow part of the soil, where phosphate tends to be more abundant and thus maximizing its uptake. This process is under the control of spatiotemporal-regulated gene expression networks . These d COLDAIR , which i species , includiThis review considers the remarkably diverse roles lncRNAs play in environmental regulation of root system architecture.cis-NATs) or from distinct genomic loci  tail. Plant specific RNA polymerases IV and V (pol IV and pol V), which evolved from pol II (Most of lncRNAs identified in eukaryotes are likely to be transcribed by RNA polymerase II (pol II) in a process in plants involving the eukaryotic multi-subunit protein complex Mediator . They alm pol II , also acm pol II , whereasm pol II .7mGTP-capped, but do not have 3′-poly(A) tails  tails . A compaA) tails .Arabidopsis uncovered nearly 6,500 lincRNAs that accumulate across diverse organs of Arabidopsis, of which 2,708 were reconfirmed in a subsequent RNA-seq analysis , which encodes a regulatory kinase that determines the polar localization of the auxin transporter PIN-FORMED 2 (PIN2) in root cells , a member of the Polycomb Repressive Complex 1 (PRC1), whereas pol IV/V transcription of APOLO triggers its methylation, mediating loop closure and thus downregulation of PID transcription by pol II. Hence, this mechanism fine-tunes PID promoter activity and could enable oscillation of gene expression in roots in response to auxin.By use of a system that allows synchronization of the formation of lateral organs in roots, the authors showed that auxin recruits the DNA Demethylation Machinery (DDM) at the Arabidopsis  of a specific subset of genes which are involved in the control of lateral root initiation  was identified in a screen for proteins that bind the short open reading frame mRNA called ENOD40, a highly structured long RNA . This study found ENOD40 overexpression caused the relocalization of MtRBP1/MtNSR from nuclear speckles to the cytoplasm. Interestingly, this phenomenon was recapitulated when ENOD40 was co-expressed with AtNSR in tobacco cells. An Arabidopsis lncRNA that binds AtNSRs called ASCO lncRNA (AS competitor long non-coding RNA) was identified  of bidopsis . The parn Figure . In a prentified . The strPlants have evolved an array of physiological and biochemical responses to cope with dynamics in the moisture, nutrients and microbiome of the soil. This is particularly striking when plants experience limiting Pi availability, which promotes the root system to undergo profound changes in morphology and architecture. This is accompanied by metabolic reprogramming to maintain intracellular Pi homeostasis.At4 and IPS1 (INDUCED BY PHOSPHATE STARVATION 1) and their orthologous in Medicago (Mt4) and tomato (TPS1) respectively, are strongly induced by Pi starvation. Loss-of-function mutation of At4 results in defects in Pi redistribution between root and shoot under Pi deficient conditions  was shown to act as a translational enhancer of OsPHO1;2 and to contribute to Pi homeostasis  regulates UCHL1 synthesis without changes at the transcript level (Uchl1 mRNA. However, PHO1;2cis-NAT does not have features of a SINE or other repetitive element. Nonetheless, subcellular transcriptomic analysis in human cell lines revealed a large fraction of lncRNAs is enriched in the cytosol as well as polysomal fractions, whereas only a small subset was found in the nucleus (Arabidopsis included about 10% of the annotated lncRNAs (It was hypothesized that s Figure . Stimulapt level . These a nucleus . Nucleas lncRNAs , with moThe recent burst of papers describing roles of lncRNAs in plant development and environmentally-regulated developmental plasticity highlights the need for deeper study of the contributions of lncRNA to dynamics of gene expression networks. This is particularly relevant in the control of the root system response to changes in nutrient and water availability where fine-tuning of developmental regulator genes is required to trigger the rapid and reversible developmental and physiological alterations needed to obtain moisture and nutrients.Functional analysis of lncRNAs is challenging. In many cases, their activity may rely only on secondary structure rather than primary nucleotide sequence. However, the recent advances in genome engineering using designer site-specific nucleases such as CRISPR/Cas9 and TALENs, as well the ability to access to cell-specific and/or subcellular RNA populations using technologies such as isolation of nuclei tagged in specific cell types INTACT;  and tranThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


AbstractObesity, defined as a body mass index (BMI) >30 kg/m2, has seen an important increase in prevalence in the last decades, not only in Europe and the United States, but also in developing countries. It is an established risk factor for numerous pathologic conditions like diabetes mellitus, cardiovascular diseases and cancer, but has also been linked to male hypogonadism. Several studies showed a negative impact of excessive BMI on testosterone levels, sexual function and sperm parameters. Possible mechanisms beyond this phenomenon are reduced hypothalamic and pituitary secretory function, excess estrogen production and reduced circulating sex-hormone binding globulin (SHBG). Peptides produced by the adipocyte may also trigger modifications of the reproductive function. Independently of the method used, non-surgical approach or bariatric techniques, weight reduction and a return to a normal BMI have been associated with improvement in the sexual function and levels of sexual hormones in obese males, showing that obesity related hypogonadism is preventable. Sexual and reproductive health might represent additional motivational factors for men in order to maintain a healthy life-style. In 2008, about 35% of the adults above the age of twenty were overweight and 11% were obese [1]. Initially considered a condition typically found in high-income societies, obesity turned epidemic also among the developing countries. As a result, more than half of the world's population lives in countries where overweight and obesity have a higher impact on mortality than underweight [1]. The growing prevalence of obesity may be attributed to increased intake of high-caloric food and decreased physical activity, common features of the modern lifestyle. Obesity is therefore the result of an imbalance between caloric intake and energy expenditure.In this new century, obesity and overweight represent a major matter of public health. According to the World Health Organization, the worldwide prevalence of obesity has almost doubled since 1980 [2]. Although the consequences on human reproduction are still under discussion, studies on adult males report an association between obesity and impairment of reproductive hormones or sperm parameters [3]. The aim of this study is to explore the impact of obesity on the male reproductive system, to review the current mechanisms proposed, and to discuss the prevention strategies.  Obesity has a clear impact on the onset or progression of various diseases like type-2 diabetes, hypertension, osteoarthritis and even cancer [ Definition of obesity 4]. Calculating BMI, the ratio between the body weight in kilograms and the height in meters squared, remains the main method to identify obese patients in both men and woman. The World Health Organization considers overweight a BMI >25 kg/m2 and obesity a BMI >30 kg/m2 [1].  The most sensitive methods of quantifying human adipose tissue are radiologic: dual energy X-ray absorptiometry, computed tomography or nuclear magnetic resonance. Less precise methods, but easier to use in clinical practice, are anthropometric measures like body mass index (BMI), waist circumference and waist-hip ratio. For men, a waist circumference >102 cm or a waist–hip ratio >0.9 indicate increased body fat and are considered predictors of the occurrence of comorbidities [ Overview of the male reproductive system5]. It triggers the release of Gonadotropin-Releasing Hormone (GnRH) pulses from the hypothalamus that further stimulates the secretion of gonadotropins (Follicle-Stimulating Hormone [FSH] and Luteinizing Hormone [LH]) by the anterior pituitary. FSH and LH act at the testicular level to induce sperm maturation at the tubular compartment and steroid hormone production at the interstitial compartment. More than 95% of the male androgen hormones derive from the testis and testosterone is the main product of the Leydig cell, with a daily normal output of 6-7 mg [6]. Testosterone has a pivotal role in spermatogenesis and in fertile men testicular concentrations are >80-fold higher than blood concentrations. The serum total testosterone is of 44% bound to Sex Hormone Binding Globulin (SHBG) and 54% to Albumin, only about 2% representing the free ratio [6]. Testosterone has a key role in triggering male sexual characteristics such as the development of genital organs, muscle and bone mass, and body hair growth. Part of the androgenic effects reflects a direct action of testosterone on target cells, whereas others are the consequence of the peripheral conversion to 5α-dihydrotestosterone and estradiol.  The reproductive function starts with the signal provided by the hypothalamic peptide Kisspeptin [ Impact of obesity on the male reproductive axis: clinical evidence7]. Whereas male infertility often triggers the lack of conception, symptoms and signs of androgen deficiency are variable because they can be modified according to age, duration of deficiency, concomitant diseases, replacement therapy or individual perception. Some clinical features of hypogonadism are highly specific , whereas others are less  [7]. Male hypogonadism is a syndrome that results from the inability of the gonads to provide physiological levels of testosterone and a normal number of spermatozoa  due to an impairment at one or more levels of the hypothalamic-pituitary-testicular axis [3]. Among 390 men from infertile couples but without a known male infertility factor, Hammoud et al. [8] found an odds ratio (OR) for oligozoospermia of 3,3  for obese males compared to patients with normal BMI. Furthermore, the OR of having a progressively motile sperm count <10x106 was 3,4 . Hofny et al. [9] made a comparison between 42 fertile obese and 80 infertile obese men and found a significant difference in BMI between the two groups , BMI having a significant negative correlation with sperm count and sperm motility. Stewart et al. evaluated a cohort of 225 Australian fertile men and showed that men with BMI >30 kg/m2 had a significantly lower total sperm count than those with BMI <30 kg/m2 [10]. A recent meta-analysis that included 14 studies corresponding to 9779 male subjects concluded that obese men had a higher risk of oligozoospermia  or azoospermia  compared to normal-weight men [11]. On the contrary, the cross-sectional population-based study involving 1989 men conducted by Aggerholm et al. did not show any difference in sperm concentration and total sperm count between the obese group and the normal weight group (68 x 106 /ml [95% CI 58-78]) versus 59 x106 /ml [95% CI 55-63], and 190 x106 /ml [95% CI 161-223] versus 168 x106 /ml [95% CI 157-180]) [12]. In line with the above results, the meta-analysis conducted by MacDonald et al. concluded that evidences do not show a clear relationship between BMI category and the mean or median sperm concentration, mean or median total sperm count, mean semen volume and average sperm motility [13]. While several studies suggest a link between increasing BMI or waist/hip ratio and impairment of seminal parameters, a similar group of studies does not [14]. Most studies report a reduction in plasma SHBG and total testosterone levels, whereas a weaker relationship is observed between BMI and free testosterone. Estradiol levels can be elevated in obese patients, but MacDonald et al. found a positive relationship with BMI only in four out of the ten studies measuring estradiol [13]. As reviewed by Teerds et al., in 88% of the analyzed studies, the decrease in testosterone was not parallel to a reduction in LH and FSH levels and obese males often showed normal gonadotropin levels [3]. Erectile dysfunction and reduced libido are also common causes of sexual dysfunction among obese males. Despite the differences in the geographical area of setting and heterogeneity of evaluation methods, a number of eight population based studies showed a higher prevalence of erectile dysfunction in obese males compared to normal-weight subjects [15]. To bring an example from the European population, the survey “Contexte de la Sexualité en France" that included 4635 men, found a higher occurrence of erectile dysfunction in obese males  [16].  A significant body of evidence makes the relationship between obesity and impairment of reproductive hormones or presence of symptoms of hypogonadism less debated [ Possible mechanisms beyond the negative effect of obesity on the male reproductive axis 17]. The exact mechanisms underlying the influence of obesity on the male reproductive axis in humans are still under debate. A normal energy balance consists of sufficient caloric input, adequate energetic expenditure and efficient thermoregulatory costs. Each level of the reproductive axis  is able to respond to the energetic modifications. In cases of long-term impairment of the energetic balance, the pulsatile secretion of GnRH is modified and subsequent long term effects on the male reproductive function can occur [18]. The negative correlation between BMI and SHBG might be the result of hyperinsulinism, because insulin has shown to be an important inhibitor of hepatic SHBG output [19]. Furthermore, plasma SHBG levels return to normal when weight loss is achieved [20]. An important issue debated today is whether testosterone and SHBG reduction are clinically evident. If the testicular testosterone output is severely reduced and not parallel to the reduction of SHBG, free testosterone levels could probably also show a decrease [3].  As previously stated, total testosterone is often reduced in obese males. Presumably, this imbalance is, at least in part, explained by the reduction in blood SHBG levels, frequently found in obese subjects [22]. An increased testosterone/estradiol ratio has been found to be linked to male infertility [23]. Visceral adiposity could therefore act as an endocrine-disruptor, reducing gonadotropin secretion through a negative feedback of estradiol at a hypothalamic and pituitary level, establishing a condition of hypogonadotropic hypogonadism [24]. This phenomenon probably occurs only in a subgroup of patients, as supported by the rich number of studies that showed normal gonadotropin levels in obese males [3]. Excess adipose tissue might be the cause of increased aromatization of testosterone, resulting in higher estradiol levels [14]. Leptin is a 16 kDa polypeptide produced mainly at the adipocyte level but also at the hypothalamic, pituitary, gastric and gonadal level, with important functions in the energetic balance of the organism [25]. Leptin acts as a satiety signal and levels increase parallel to weight gain, being a good parameter of body adipose stores [26]. An effect on GnRH and LH pulsatile secretion is presumed, as demonstrated by the association with puberty onset [27]. In human males, leptin was found negatively correlated to total and free testosterone, also after adjusting for the effect of SHBG, LH and estradiol, resulting a good predictor of low androgen levels in obese subjects [28]. Adiponectin is a polypeptide produced exclusively at the adipose level, that reduces hepatic glucose production and increases hepatic insulin sensitivity . Obese subjects often show low adiponectin levels, negatively correlated to insulin resistance and risk of type-2 diabetes [30]. Adiponectin is thought to exert a permissive effect on LH release because its receptors were found at the hypothalamic and pituitary level [31].  A number of at least 30 peptides are produced by the white adipose tissue, most of them with biological effects on the human organism. Some of them are related to the reproductive function in animal models, most likely because of an action at the hypothalamic level [32]. Studies that are more recent have advanced the hypothesis that oxidative stress and lipid peroxidation could be the link between genital adiposity and infertility in obese males . Sleep apnea, a condition characterized by frequent proximal airway tract obstruction resulting in apneas, hypopneas, oxygen desaturation, and arousals from sleep, is frequently associated with the obesity hypoventilation syndrome [35]. A reduction in LH and testosterone, as well as an increase in erectile dysfunction prevalence has been found in obese males that present sleep apnea [36-38]. Obesity is associated to mechanical factors that have been linked to impairment of the male reproductive function. Excess suprapubic adipose tissue has been initially considered a cause of increased scrotal temperature and a negative effect on spermatogenesis could be the possible consequence [ Consequences of weight loss 39]. Therapeutic interventions used in surveys that evaluated male androgen status included surgery and lifestyle changes . The above presented evidences show that obesity has an impact on the male reproductive axis and could trigger infertility or androgen deficiency. Most authors agree that the first step in approaching this condition should be the weight reduction, demonstrating that obesity related hypogonadismais preventable. Only few drugs are approved today in the United States for the treatment of obesity , but none of them has been used yet in male patients with low androgen levels or symptoms of hypogonadism [40]. A randomized single-blind trial that delivered a twenty-four months detailed training on caloric intake and physical activity to 55 obese men with erectile dysfunction showed a higher degree of weight loss and a significant improvement in erectile function scores compared to the control group that received only general information about healthy food choices and physical activity [41]. Bariatric surgery, a treatment indicated for morbid obesity (BMI>40 kg/m2 or >35kg/m2 with comorbidities), has shown to provide consistent weight loss and reduction in comorbidities [42]. A recent meta-analysis that evaluated the consequences of weight loss strategies on androgenic environment showed that all the surgical techniques have been followed by an increase in the levels of testosterone , SHBG and gonadotropins [43]. After nine weeks of a very low-calorie diet, the 58 obese men evaluated by Niskanen et al. showed a significant increase in SHBG, total and free testosterone, a trend that has also been maintained after a twelve months of follow-up [Obesity is a complex medical condition associated with chronic pathological conditions that have an increased risk of mortality. Important modifications in the quality of life are also commonly encountered in obese subjects, and part of them could be related to the sexual and reproductive dysfunction. A significant group of evidences presented to date showed that obese males are more likely to develop androgen deficiency and reduced fertility. On the other hand, cohorts of obese subjects do not report symptoms of hypogonadism and have fathered their own offspring. A possible explanation of this disagreement is that a higher testicular output of testosterone, as seen in young adult males, could maintain a normal androgenic activity. Furthermore, female fertility could compensate the impairment in sperm parameters, providing the achievement of a pregnancy. Therefore, data available today seem to argue that obesity represents a risk factor for male sexual and reproductive dysfunctions. Improvement in reproductive hormones are expected after reducing body fat mass and this finding should be a further motivational factor to induce obese men to lose weight.


Where biological datasets are spatially limited, abiotic surrogates have been advocated to inform objective planning for Marine Protected Areas. However, this approach assumes close correlation between abiotic and biotic patterns. The Solitary Islands Marine Park, northern NSW, Australia, currently uses a habitat classification system (HCS) to assist with planning, but this is based only on data for reefs. We used Baited Remote Underwater Videos (BRUVs) to survey fish assemblages of unconsolidated substrata at different depths, distances from shore, and across an along-shore spatial scale of 10 s of km (2 transects) to examine how well the HCS works for this dominant habitat. We used multivariate regression modelling to examine the importance of these, and other environmental factors , in structuring fish assemblages. There were significant differences in fish assemblages across depths, distance from shore, and over the medium spatial scale of the study: together, these factors generated the optimum model in multivariate regression. However, marginal tests suggested that backscatter intensity, which itself is a surrogate for sediment type and hardness, might also influence fish assemblages and needs further investigation. Species richness was significantly different across all factors: however, total MaxN only differed significantly between locations. This study demonstrates that the pre-existing abiotic HCS only partially represents the range of fish assemblages of unconsolidated habitats in the region. To adequately represent the entire range of biota present, conservation planning for sub-tidal marine ecosystems should be based on comprehensive understanding of species, habitats, ecosystems and associated ecological processes Sub-tidal sediments are the most extensive benthic habitat worldwide Most data on demersal fish assemblages of unconsolidated habitats have been collected by trawling The Solitary Islands Marine Park (SIMP) in the Tweed-Moreton Bioregion of northern New South Wales, eastern Australia, comprises both State waters , and the adjacent Solitary Islands Marine Reserve (SIMR) in Commonwealth waters. The SIMP is unique among NSW marine parks in that it includes several islands up to 12 km from the mainland coastline. The dominant system of currents, which includes strong influence of the East Australian Current (EAC) Unconsolidated sediments dominate sub-tidal habitats in the SIMP The focus of this research, therefore, was to examine fish assemblage structure of unconsolidated habitats to determine patterns over factors known to affect their reef counterparts , and to determine how consistent they are over medium spatial scales (km). As data on the broad composition of unconsolidated habitats has recently been generated using swath acoustic multi-beam sidescan sonar The SIMP covers an area of approximately 71,000 hectares, within a broad ecotone between tropical and temperate assemblages Sardinops neopilchardus) was mashed into the bait bag, approximately 1.5 m from the lens of the camera, to attract fish to the viewing area in front of the camera. Each housing and bait pole was bolted to the attachment frame so that fish could be viewed in a horizontal orientation to the substratum. The field-of-view was standardised to approximately 2 m behind the bait, to minimise the effects of water clarity on measures of relative abundance Each BRUV drop consisted of a mini-DV video camera with a wide angle lens, within an underwater housing with flat acrylic end ports, an attachment frame, a bait pole and mesh bag with bait, and a rope and float system linking the unit to the surface Files were analysed using Eventmeasure , and the identity and MaxN of each fish species was recorded. MaxN is the maximum number of fish of each species within the field-of-view at any one time during the 30-min recording, which removes the possibility of recounting the same fish. As counts reflect relative abundance and not density, we expect our data to be robust to variability in observing the field-of-view The placement for all BRUV deployments was determined using GIS high-24 resolution imaging software  which incorporated metrics for the factors of primary interest: depth; distance from shore; and along-shore location (as distance from latitude -29 S – hereafter termed location). Additional factors, including fine-scale bathymetric range, rugosity and backscatter were also considered. Bathymetry and backscatter data (5 m cell size) were provided by the NSW Office of Environment and Heritage (2012). Rugosity was derived from the bathymetry layer using the Benthic Terrain Modeller extension 1.2 2 and Akaike Information Criterion (AIC) Both multivariate and univariate analyses were performed using procedures in the PRIMER 6.0 software Distance based redundancy analysis (dbRDA) biplots were generated to visually display the direction and magnitude of the relationship between habitat factors and individual fish species Univariate permutational multivariate analysis of variance (PERMANOVA) (using Euclidean distance as the similarity measure) was performed to test for significant differences in Species Richness and Total MaxN between factors that were significant within the DistLM regression. Species Richness is calculated as the total number of different species viewed on each replicate video, and Total MaxN is calculated as the sum of MaxN for all species per replicate. Differences across bathymetry and rugosity categories were not analysed as the range of values was very low.post-hoc pair-wise contrasts were performed to establish where differences in assemblage structure were occurring.One-way PERMANOVA Multivariate assemblage structure was visually examined using non-metric multi-dimensional scaling (nMDS) ordination. Data were square-root transformed, and a dummy variable was added, prior to the generation of Bray-Curtis similarities. The resultant nMDS was used to examine differences in assemblage structure across factors that were significant in the DistLM analysis. Depth-related patterns were visually examined to see how well they fitted the current depth-based habitat classification categories Platycephalus caeruleopunctatus, P. longispinis and Aptychotrema rostrata. Schooling Sillago spp. were numerically abundant in some positions, and recorded across all depths, but were highly variable between positions and between depths.A total of 16 teleost and elasmobranch species, from 12 families, was recorded from BRUVs deployments. Platycephalidae was the most speciose family (3 species). Three species were numerically abundant and ubiquitous across all BRUV positions in the study – 2 and AIC selection criteria. However, the optimum model for adjusted R2 included all four factors: for AIC, only depth and location were included in the optimum model. The adjusted R2 model explained 23.1% of the total variation, while the AIC model explained 19.8%  correlated closely with the first dbRDA axis, with backscatter intensity also partially correlated (ρ = 0.62). The second axis was a combination of location (ρ = −0.85) and depth (ρ = −0.42). For the AIC model depth (ρ = −0.99) was almost perfectly correlated with the first axis, while location (ρ = −0.99) was similarly correlated with the second axis.Marginal tests in distance-based linear modelling (DistLM) showed that, of the four environmental factors retained for analysis, three were significant in isolation  for bothed 19.8% , Table 22 model must be interpreted somewhat cautiously, as the first dbRDA axis represents a combination of depth and backscatter, while the second axis is a combination of location and depth. Therefore, determination of the factor primarily responsible for the distribution of each species is partially confounded. Some of the species and their respective correlation values for both the adjusted R2 and AIC models are displayed in By examining the raw Pearson correlations for different species, we could identify those most responsible for driving the assemblage response to each environmental factor: these are displayed as vectors in Species richness was generally higher in the south of the park . The higPERMANOVA revealed a significant difference between species richness for all four factors of interest . PairwisTotal MaxN values were slightly higher in the south of the park, and showed trends similar to those seen for species richness . The higPERMANOVA revealed significant effects for all three selected factors . PairwisSome trends were evident in the nMDS ordination  with thePatterns of fish assemblage structure in unconsolidated habitats within the SIMP are clearly influenced both by depth and factors operating over medium spatial scales (i.e. the distances between our locations). Additionally, backscatter intensity, while not a primary driver, was highlighted as potentially explaining some of the differences in fish assemblages across the scales of the study.Worldwide, fish assemblage patterns of both reef and unconsolidated habitats have often been found to be correlated with depth The importance of depth in structuring assemblages reflects what is known from other habitats in the SIMP and elsewhere. Strong depth-related patterns have been detected in reef fishes With strong co-linearity between depth and distance from shore within our study, it is difficult to distinguish which factor primarily drives the observed patterns. Shelf position is included in the current HCS for the SIMP, as distinct cross-shelf patterns have been demonstrated for corals The correlation with location in our modelling contradicts findings for reef fish communities in the SIMP Pseudorhombus arsius, Parapercis stricticeps, Centropogon australis, Ratabulus diversidens) were found only at sites with a backscatter index ≥50, despite a low number of replicates with this index or greater (15) relative to the total  in the distribution of different species. With obvious and extensive differences in sediment characteristics, it is likely that, in addition to the other factors identified in this study, sediment type and habitat complexity are important factors structuring fish assemblages. Recent research in the SIMP Our modelling also suggests the possibility that sediment grain size, for which backscatter intensity is an appropriate surrogate, may influence fish communities of unconsolidated habitats. Broad-scale bathymetric mapping of the seafloor within the SIMP has shown that unconsolidated habitats show distinct variability in sediment type, as revealed in acoustic backscatter  65, see . AdditioWhen compared to reefs, unconsolidated habitats, particularly in a high energy environment, have lower topographical complexity, support fewer sessile biota, and provide limited foraging opportunities and protection from predation The current Habitat Classification System (HCS) for the SIMP has been developed and adjusted primarily based on fish assemblage patterns associated with hard substrata The availability of high resolution benthic mapping has allowed us to examine the influence of a range of factors, which would otherwise be difficult and expensive to assess, on the poorly described fish assemblage of unconsolidated habitats. The patterns evident within the study endorse the relevance of depth and distance from shore as categories currently used as biophysical surrogates to represent discrete assemblages in marine parks in NSW. However, the study also indicates that the inclusion of habitat type, based on backscatter and possibly other remotely-sensed metrics, will lead to better representation of assemblages of unconsolidated habitats.


Here, we review the characteristics, regulation, in vitro derivation and in vivo induction of NMps. We propose that these cells arise within primitive streak-associated epiblast via a mechanism that is separable from that which establishes neural fate in the anterior epiblast. We thus argue for the existence of two distinct routes for making central nervous system progenitors.Neuromesodermal progenitors (NMps) contribute to both the elongating spinal cord and the adjacent paraxial mesoderm. It has been assumed that these cells arise as a result of patterning of the anterior neural plate. However, as the molecular mechanisms that specify NMps  The vertebrate central nervous system (CNS) is first manifest as an ovoid region of thickened epiblast cells in front of the organiser/anterior primitive streak. This region is known as the anterior neural plate . Fate-maThe prevailing view of vertebrate neural induction derives largely from work in the amphibian embryo. This proposes that initial induction of the anterior neural plate is followed by the formation of more posterior neural regions via patterning of this anterior tissue with posteriorising signals  . This viThe molecular basis for this ‘activation’ step is not without controversy when extended to amniote embryos. Although inhibition of BMP signalling promotes neural fate in the mouse embryo, for example , BMP inhin vitro  that contributes to both the spinal cord and paraxial mesoderm in the mouse embryo  has now in vitro , raisingin vitro . These cpulation , lendingClearly, the existence of NMps challenges traditional notions of the formation of three germ layers  and subsequent neural cell fate assignment from within the ectoderm. In the prevailing view of neural induction, NMps are derived from the anterior neural plate, and the setting aside of these cells from within this neuroepithelium might then be considered a patterning event dependent on prior formation of anterior neural tissue . An altein vivo and in vitro. We also evaluate experiments in the embryo, which suggest that anterior and posterior neural tissue can form independently. Finally, we review lineage data and gene regulatory interactions to speculate on the point at which anterior-posterior pattern and neural fate are established in the early epiblast and how this relates to the induction of NMps.Here, we review the evidence for NMps, focusing largely on data from amniote embryos, and consider their molecular characteristics and the signals that induce them laacZ transgene reverts at low frequency to a functional lacZ gene, the expression of which marks the single revertant cell and all its progeny (constituting a clone)  . The anaa clone) . This sua clone) . Indeed,a clone) . One intRetrospective clonal analysis does not directly indicate the location of NMps in the embryo. However, fate-mapping studies in which small groups of cells were labelled have helped to identify regions where NMps may reside in the embryo. In the chick, dye labelling of groups of one to three cells in the CLE identified a region close to the primitive streak that is able to contribute to both neural and mesodermal lineages at early somite stages . Labelliin vitro differentiation (discussed in detail below). Recent work using this approach also provides evidence strongly suggesting that single cells with the molecular hallmarks of NMps can give rise to clones containing both neural and mesodermal progenitors (Further persuasive evidence for the existence of NMps comes from the ability to derive cells with these characteristics from pluripotent stem cells via the approach of genitors .Sox2 has been used to identify these cells in the epiblast associated with the primitive streak (Bra-expressing cells (using Bra-Cre lines) has indicated that these cells contribute significantly to the spinal cord (see below), confirming that Bra is indeed expressed in cells with neural potential, in addition to its well-known expression in prospective mesoderm  and the neural progenitor marker e streak . In mouse streak . Furthermesoderm .Sox2 expression is driven by a unique enhancer element (termed N1), which, importantly, is distinct from that (N2) promoting Sox2 expression in ESCs and subsequently in the anterior epiblast    and the )  (Cash4  and thus) (Cash4 . Dye lab)  and inhibits the progression of differentiation in this caudal region  is also inhibited by BMP signalling, which restricts Sox2 transcripts to the CLE/NSB , which are key mediators of caudal Hox gene expression  was crucial to generate NMps , most likely induced by activin. Gene expression analysis confirmed activation of the Wnt pathway by CHIRON99021 and the upregulated expression of various lineage-affiliated genes, including endodermal, mesodermal and neural markers, together with a strong repression of the pluripotency genes Oct4 (Pou5f1) and Nanog. In addition, known anterior neural markers such as Pou3f2 were repressed, whereas posterior markers  were induced.An initial report  describeBra-expressing cells in vivo. However, as in the embryo, it is not simply the case that maintenance of Wnt signalling promotes mesodermal over neural fate in this context. For example, Bra null ESC-derived NMps exposed to CHIRON99021 fail to make mesoderm, but can still form neural tissue. This is consistent with findings in the embryo that Wnt signalling is not incompatible with the generation of neural fates from NMps. The apparent multiple roles of Wnt signalling in caudal tissues require further investigation, and this new ability to generate NMps in vitro will now permit precise investigation of Wnt signalling in the control of NMp specification, maintenance and differentiation.Subsequent work demonstrated that the exposure of both mouse and human ESCs to FGF2 and CHIRON99021, in the absence of activin, led to more efficient generation of NMps, reaching up to 80% of the cells in culture ; a regimin vitro generated NMps and, as noted above, this has been followed up by data which strongly suggest that single Bra/Sox2 co-expressing cells can generate clones containing neural and mesodermal cell types in vitro . Each population also deployed different FGF ligand-receptor combinations, with NMps expressing Fgf4, Fgf8 and the receptor Fgfr1, and anterior precursors expressing higher levels of Fgf5, 14 and 15, and of Fgfr2 and 3. The two populations also appear to use distinct mediators of BMP inhibition; anterior neural precursors express higher levels of Smad7, whereas neural precursors derived from NMps have higher levels of Smoc1. A further distinguishing feature is the response to RA signalling, which promotes hindbrain and anterior spinal cord fates in anterior neural precursors, whereas neural precursors derived from NMps acquire more posterior spinal cord fates, expressing more 5′ Hox genes  achieved by addition of the TGFβ ligand GDF11. Moreover, when NMps at different time points were exposed to RA, they downregulated BRA expression, entered neural differentiation and generated motoneurons with anterior-posterior identities according to the combination of Hox genes expressed at the time of RA addition. These findings thus support the model deduced from work in the embryo in which exposure to RA inhibits FGF/Wnt signalling and so arrests the temporal progression of 3′ to 5′ Hox gene expression, thereby setting the Hox code as differentiation commences Bra-Cre-bsomite 6  and thatsomite 6 . FurtherBra-Cre-expressing cells are found in ‘trunk neural tube’ sections Otx2 is fNkx1.2 but repress Bra in EpiSCs  and Sox2 N2 (in anterior epiblast) enhancer activity. In the primitive streak, onset of N1 activity occurs as Otx2 is downregulated, and is promoted by FGF and Wnt signalling, whereas in the anterior epiblast there is a switch to Otx2-dependent Sox2 N2 activity. (3) NMp induction appears to be linked to primitive streak induction, as ectopic FGF can induce streak-like structures that include Cash4/Bra co-expressing cells. This conclusion is further supported by studies of the in vitro induction of NMps elicited by FGF and Wnt signalling, which would be provided by the primitive streak in the embryo.This NMp induction step appears separable from that of anterior neural plate induction, for the following reasons. (1) Anterior neural plate and NMp lineages diverge at late primitive streak stages prior to the establishment of neural fate in the epiblast. (2) The molecular mechanisms for making NMps are distinct from those that direct anterior neural plate; this is indicated by the different inputs that promote Once established, NMps serve to extend the generation of new neural tissue until the end of body axis elongation, long after the node has lost its neural inducing ability, producing new neural progenitors that fuel the CLE. The production of neural and mesodermal tissue from this common precursor might then help to coordinate the differentiation and patterning of trunk tissues, as signals, such as RA, from the differentiating mesoderm then act back to repress FGF and Wnt signalling and promote the progression of neural differentiation .in vitro will allow researchers to dissect more finely the molecular mechanisms that direct neural and mesodermal differentiation and will facilitate biochemical and genome-wide approaches, such as RNA-seq and ChIP-seq, that are currently challenging in embryonic cell populations. Finally, the in vitro generation ofNMps further opens up the possibility of investigating these processes using human pluripotent cells and exploring the potential therapeutic use of NMps (Altogether, these findings suggest that there are then two routes for making CNS neural progenitors: one involves the induction of the anterior neural plate and a second the induction of NMps in the primitive streak-associated epiblast, with a subsequent ongoing decision between neural and mesodermal fates. It will be interesting to determine what is shared and what is distinct about the molecular mechanisms that generate neural progenitors via these different routes. Further important questions are raised in this advancing area of research see . In addi of NMps .http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.119768/-/DC1Supplementary material available online at Figure S1Table S1


The cyanobacterial thylakoid membrane represents a system that can carry out both oxygenic photosynthesis and respiration simultaneously. The organization, interactions and mobility of components of these two electron transport pathways are indispensable to the biosynthesis of thylakoid membrane modules and the optimization of bioenergetic electron flow in response to environmental changes. These are of fundamental importance to the metabolic robustness and plasticity of cyanobacteria. This review summarizes our current knowledge about the distribution and dynamics of electron transport components in cyanobacterial thylakoid membranes. Global understanding of the principles that govern the dynamic regulation of electron transport pathways in nature will provide a framework for the design and synthetic engineering of new bioenergetic machinery to improve photosynthesis and biofuel production. This article is part of a Special Issue entitled: Organization and dynamics of bioenergetic systems in bacteria, edited by Conrad Mullineaux. •Cyanobacterial thylakoid membranes carry out both oxygenic photosynthesis and respiration.•Electron transport components are located in the thylakoid membrane and functionally coordinate with each other.•Distribution and dynamics of electron transport components are physiologically regulated in response to environmental change. They were not only among the early microbial pioneers of life to produce oxygen that is indispensable for sustaining aerobic life in the atmosphere, but also have consistently served as a predominant contributor to our sustainable environment for around 3.5 billion years This review focuses on the spatial organization and dynamics of electron transport chains in cyanobacterial thylakoid membranes, and discusses the functional significance of the distribution and mobility of electron transport modules in vivo. The dynamic modulation of the electron flow network produces optimized energy transduction in cyanobacteria. Extensive study of cyanobacterial photosynthetic membranes will provide essential information for the design and engineering of new photosynthetic machinery and devices, with the attempts to improve bioenergy production. In addition, given the close evolutionary relationship between cyanobacteria and chloroplasts, the photoheterotrophic cyanobacteria represent an important model for elucidating the structure and function of chloroplasts in higher plants. Alternatively, knowledge obtained from plant chloroplasts will also inform the study of cyanobacterial thylakoid membranes.22.1Synechocystis sp. PCC6803 (hereafter Synechocystis 6803). The photosynthetic electron transport complexes in the thylakoid membrane include phycobilisome (the membrane associated antenna complex), photosystem II (PSII), photosystem I (PSI), cytochrome (cyt) b6f and ATP synthase (ATPase). In addition, there are small electron transport molecules, such as plastoquinone (PQ), plastocyanin (PC) and cytochrome c6, functioning as electron carriers to shuttle electrons between each electron transport complex and functionally link all the complexes together A unique structural feature of cyanobacterial thylakoid membranes is that it harbors the elements of both photosynthetic and respiratory electron transfer chains, and thereby is capable of performing both oxygenic photosynthesis and aerobic respiration in the same cellular compartment. The major photosynthetic and respiratory electron transport complexes have been structurally characterized in the past decade. a in photosystems (major absorption at 440 nm and 680 nm). Moreover, stepwise energy transfer within the phycobilisome could also act as a photoprotective mechanism to prevent the photodamage of photosystems by excess light energy b6f and PC. PSI catalyzes the light-driven electron transport including the oxidation of luminal electron carriers, PC and cyt c6, and the reduction of ferredoxin. The electron transfer reactions are coupled with the formation of an electrochemical gradient across thylakoid membranes, which is essential for driving ATP synthesis by the ATPase. This electron flow pathway, namely the linear electron transport, is strictly correlated with the evolution of O2. During the last steps of electron transfer, the ferredoxin, a strong reductant, transfers electrons to ferredoxin-NADP+ oxidoreductase (FNR) to generate NADPH. Apart from the linear electron transport, PSI also participates in the cyclic electron transport, which generates only ATP without any accumulation of NADPH, in order to balance the ratio of ATP and NADPH in the cell 2 fixation and other cellular metabolism.Cyanobacteria have evolved the extrinsic supramolecular phycobilisomes associated to the cytoplasmic surfaces of thylakoid membranes, serving as the major antenna for both photosystems O) and red forms (OCPR) R binds to the phycobilisome core, where it takes excitation energy from phycobilins and converts it to heat as an energy quencher, in order to prevent photodamage of reaction centers at high light. The reversal of OCP-induced energy quenching (conversion of OCPR back to OCPO) depends on a second cytoplasmic protein, the fluorescence recovery protein, which binds to OCP and weakens its association with phycobilisomes Likewise, some protein complexes are also related to the photosynthetic electron flow. A water-soluble orange carotenoid protein (OCP) was shown to mediate directly the fluorescence quenching of phycobilisomes, known as non-photochemical quenching, and possibly in the regulation of energy transfer between phycobilisomes and photosystems b6f2 uptake b6f and the luminal electron carrier, PC or cytochrome c. Afterward, they could be transferred to either a terminal oxidase to perform conventional respiratory electron transport with net oxidation of the metabolite pool, or could be transferred to PSI to participate in the cyclic photosynthetic electron transport. The redox state of PQ pool has been demonstrated to play an important role in steering the electron flow into different directions In contrast to the well-studied photosynthetic electron transport chain, the respiratory electron transport chain in cyanobacteria is much less understood. The main respiratory electron transport complexes include type-I NAD(P)H dehydrogenase (NDH-1), type-II NAD(P)H dehydrogenase (NDH-2), succinate dehydrogenase (SDH), cytochrome oxidase and alternative oxidases, as well as cyt b6f, PQ and PC/cyt c, are functionally shared by both photosynthetic and respiratory electron transport pathways The components of both photosynthetic and respiratory electron transport chains are structurally and functionally correlated with each other, ensuring highly efficient and optimized electron flow in the cell. Some of these electron transport components, for instance cyt Synechocystis 6803, based on their sequence similarity to ion channels from other species Synechocystis 6803 Gloeobacter (G.) violaceus also contains a proton-gated ion channel Despite the electron flow components discussed above, there are other components located in cyanobacterial thylakoid membranes. At least five potassium channel homologs have been identified in the genome of G. violaceus that lacks intracytoplasmic membrane in vivo G. violaceus, the cytoplasmic membrane hosts both photosynthetic and respiratory systems, which intersect and share in part common electron transfer components. Overall, co-existence of respiration and photosynthesis in the same membrane compartment indicates the close functional coordination between the two bioenergetic pathways.As shown in Synechocystis 6803 and Synechococcus sp. PCC 7942 (hereafter Synechococcus 7942), have been widely used in the laboratory research, because of their completely sequenced genomes and their superior genetic tractability. The spherical-shaped Synechocystis 6803 can be grown in several different conditions, with numerous mutants available. The thylakoid membranes in Synechocystis 6803 form curved and parallel sheets radiating out from the thylakoid organizing centers. By contrast, the rod-shaped Synechococcus 7942 possesses the thylakoid membranes that are organized in a series of regular, concentric cylinders inside the cytoplasmic membrane and surrounding the central cytoplasm. This configuration is advantageous to the study of protein distribution and mobility, especially to one-dimensional fluorescence recovery after photobleaching (FRAP) detection and analysis which is based on live-cell imaging using a confocal fluorescence microscope.Cyanobacteria are ideal organisms to investigate the processes and structures of electron transport pathways associated with oxygenic photosynthesis and respiration. In particular, two model unicellular cyanobacterial organisms, Cyanobacterial thylakoid membranes can not only produce energy, through the electron transport chains, for the cellular metabolism, but also provide the regulatory mechanisms to sense the external stimulation and modulate the cellular metabolism. Current structural model of cyanobacterial thylakoid membranes, based on mainly the atomic structures of individual protein complex and spectroscopic studies, illustrates a static view of the thylakoid membrane architecture. How the complexes and molecules are highly organized in the thylakoid membranes, in a functional context, and how they are dynamically adapted to changing environmental conditions are still poorly understood. Recent advances of analytical techniques, such as cryo-electron tomography, small angle neutron scattering, confocal microscopy and atomic force microscopy 2.22-fixing machinery located in the cytoplasm In cyanobacteria, compartmentalization appears to provide a means for metabolic reactions to be concentrated and enhanced within functional protein domains. A typical example is the proteinaceous microcompartment namely the carboxysome, the specialized COSynechocystis 6803 has been reported Synechocystis 6803: the inner thylakoid regions are concentrated with PSI, whereas phycobilisomes and PSII are preferentially located in the outer thylakoids of cyanobacterial cells Synechococcus 7942 the outer thylakoid layer contains mainly ATPase and PSI complexes, whereas PSII and cyt b6f are evenly distributed in the outer and inner thylakoid layers The compositional heterogeneity of thylakoid membrane fractions isolated from b6f or other electron transport complexes have been identified as parts of the megacomplex b6f and FNR, as well as a membrane-integral complex named PGRL1 was characterized Indeed, the occurrence of photosynthetic supercomplexes in cyanobacteria has been determined 2.3Synechocystis 6803 The biogenesis of cyanobacterial thylakoid membranes is likely to correlate with the development and processing of cytoplasmic membranes 2.4The structural flexibility of thylakoid membranes is of fundamental importance for the efficiency and regulation of oxygenic photosynthesis at the subcellular scale. Recent studies using small angle neutron scattering revealed that light could induce reversible reorganization of cyanobacterial thylakoid membranes 3The formation of functional photosynthetic apparatus is a highly dynamic process, involving de novo protein synthesis, protein self-assembly, turnover and repair, adaptive regulation and crosstalk between components. These orchestrated reactions are crucial for maintaining and optimizing the performance of photosynthetic light-energy conversion. The mobility of protein complexes in thylakoid membranes is governed by a combination of specific protein–protein interactions, membrane viscosity and macromolecular crowding. To date, our knowledge about the organization and dynamics of electron transport components in cyanobacterial thylakoid membranes is still not satisfactory. Some of the pigment–protein complexes in thylakoid membranes are naturally fluorescent, such as phycobilisomes and PSII, allowing direct visualization of the distribution and dynamics of these photosynthetic complexes in vivo without necessity for specific fluorophore binding or fluorescent protein fusion. By contrast, information about the distribution and dynamics of non-fluorescence components is very limited.3.1Synechocystis 6803 has been isolated, indicating the presence of photosynthetic antenna-reaction center supercomplex assembles in cyanobacteria Anabaena sp. PCC 7120, a phycobilisome–CpcL–PSI supercomplex was also determined Anabaena and whether such specific PSI assembly is only restricted to heterocysts. Likewise, whether the formation of these photosynthetic supercomplexes characterized is transient and dynamic in vivo remains unknown.Physical association between phycobilisomes and photosystems is prerequisite for efficient energy flow. The phycobilisome linker protein, ApcE, plays an key role in anchoring phycobilisomes with photosynthetic reaction centers  proteins Flv1–4 Porphyridium cruentum have elucidated significant macromolecular crowding of thylakoid membrane surfaces, with densely packed phycobilisomes The detailed mechanism governing the rapid phycobilisome diffusion on cyanobacterial thylakoid surfaces still remains to be explored Chlamydomonas chloroplasts As comparison, LHCs in green alga and plant chloroplasts are very dynamic. They preferentially form supercomplexes with photosystems, such as PSII–LHCII and PSI–LHCI. During state transitions, LHCII can reversibly migrate between PSI and PSII to balance the distribution of absorbed light energy 3.2Synechocystis 6803 is organized at the outer thylakoid membranes G. violaceus that has no thylakoid membranes, PSII complexes are heterogeneously distributed in its plasma membranes, and are preferentially concentrated into specific membrane domains The distribution of cyanobacterial PSII and PSI is relatively homogeneous throughout thylakoid membranes, and often both photosystems are in close proximity The biosynthesis cycles of PSII complexes are highly dynamic. In cyanobacteria, the assembly of PSII appears to commerce within cytoplasmic membranes and then carries on in thylakoid membranes It is reasonable that the diffusion of PSII assembly modules in thylakoid membranes plays a role in the dynamic life cycle and regulation of PSII. Compared to the rapid movement of phycobilisomes, however, FRAP studies revealed the membrane-integral PSII complexes are mostly immobile in cyanobacteria under low-intensity white light, probably due to the spatial constrains of macromolecular crowding and specific protein interactions in thylakoid membranes The variation of PSII mobility under distinct environmental conditions indicated the existence of specific protein–protein interactions or protein–lipid interactions that govern the functional organization of PSII in cyanobacterial thylakoid membranes. Cyanobacterial thylakoid membranes have exceptionally high protein abundance, which is an impediment to rapid protein mobility 3.3Synechocystis 6803, the ratio of PSI monomers to PSII monomers varies between 1.5:1 under light favoring PSI and 3:1 under light favoring PSII The stoichiometry of PSI and PSII in thylakoid membranes is dynamically regulated in response to light quality and quantity. In b6f, ATPase and FNR in cyanobacterial electron flow, so far there is no experimental data on the distribution and mobility of these non-florescence complexes. Our limited knowledge about the organization and dynamics of PSI, cyt b6f and ATPase is achieved mainly from the studies of bacterial membranes Though PSI is naturally fluorescent, most of the room-temperature chlorophyll fluorescence is attributed to PSII rather than PSI. This has led to very limited information about the localization and dynamics of PSI in cyanobacterial thylakoids, relative to the extensive studies on PSII. Similarly, despite the profound roles of cyt b6f complexes are more evenly distributed throughout chloroplast thylakoid membranes b6fb6f in plants b6f was detected b6f and ATPase will advance our understanding of the in vivo dynamics and functionality of these electron transport complexes in cyanobacterial photosynthetic membranes.In higher plants, PSI–LHCI and ATPases are preferentially concentrated in unstacked thylakoid regions, physically separated from PSII–LHCII supercomplexes in the stacked granal membranes. Cyt 4Synechocystis 6803 Anabaena sp. PCC 7120 Synechocystis 6803 Synechococcus 7942 are both confined exclusively in thylakoid membranes. Likewise, cyt oxidase was also found mostly in thylakoid membranes of Synechocystis 6803 In cyanobacterial thylakoid membranes, the respiratory complexes are laterally interwoven and have functional correlation with the photosynthetic complexes. The exact location of cyanobacterial NDH-1 complexes is controversial. Using different biochemical methods, NDH-1 complexes have been found in both cytoplasmic and thylakoid membranes of Synechococcus 7942 provided new insights into the large-scale reorganization and coordination of photosynthetic and respiratory electron transport pathways in cyanobacterial thylakoid membranes Our knowledge about the distribution and dynamics of respiratory electron transport complexes in cyanobacterial thylakoid membranes is fragmentary. The study of the distribution of NDH-1 and SDH complexes in Another example of dynamic “electron valves” in cyanobacteria is the Flv proteins, Flv1–4. They are cytoplasmic proteins that take electrons from the photosynthetic electron transport chain and divert them to alternative acceptors. Flv1 and Flv3 form a heterodimer that takes electrons from the acceptor side of PSI and uses them to reduce oxygen 5b6f. The redox state of PQ pool has been found to function as the key controller of a number of biological reactions in cyanobacteria, including the distribution of respiratory complexes Escherichia (E.) coli cell suspension was pre-incubated with NBDHA-Q prior to confocal imaging. This allowed the incorporation of NBDHA-Q into cell membranes, in specific the cytoplasmic membrane. There seems no PQ microdomain in the thylakoid membrane, as confocal imaging revealed relatively homogeneous halos along the cellular membrane, without any significant spotty distribution E. coli cytoplasmic membranes (E. coli cells grown at different temperatures In addition to the mobility of protein complexes, the diffusion dynamics of small electron transport molecules, comprising PQ, PC and cyt c, are also essential to electron transport embranes . The difb6f and PSI. Therefore, its mobility is largely affected by the circumstance of thylakoid lumen. It was revealed that light intensity could induce variations of the thylakoid lumenal space Another electron carrier, PC, diffuses in the lumen of the thylakoid membrane to connect cyt The organization of photosynthetic membranes plays an important role in regulating the direction and efficiency of electron flow. A common feature of all photosynthetic membranes that have been analyzed is the exceptional protein crowding 6b6f, ATPase, cyt oxidase as well as the electron carriers of photosynthetic and respiratory electron transport are imperative. In addition, little is known about the factors that govern the dynamic processes of electron transport pathways in thylakoid membranes, such as how do the lipid composition and lipid–protein interaction affect the organization and dynamics of proteins and molecules involved in electron transport. Global picture of the regulation and acclimation of the electron transport pathway network in cyanobacterial thylakoid membranes will benefit from the recent advances of “omics” approaches, including transcriptomics, proteomics and metabolomics.Evolution has developed strategies to modify, in an efficient manner, the electron transport pathways in response to diverse environments. Dynamic organization of electron transport components in cyanobacterial thylakoid membranes provides a basis for the adaptive responses that allow the regulation and optimization of both photosynthesis and respiration. Our current understanding of the functional and structural dynamics of electron transport pathways needs to be substantially improved. For example, further characterization of the dynamics of cyanobacterial PSI, cyt Advanced knowledge of the electron transport in photosynthetic membranes has led to attempts to modulate the photosynthetic building blocks and pathways, using the powerful tools of synthetic engineering. In this context, cyanobacteria can be used as an attractive “green” basis for producing high-value compounds, such as food, pharmaceuticals, and biofuels. Understanding the regulatory mechanisms underlying the larger-scale distribution and dynamics of electron transport modules will provide essential information required for developing strategies to optimize and control electron transport pathways, and bioengineering of new bioenergetic machinery or organisms to boost biofuel production.Transparency document.


A 22-year-old soldier suffered a phosphorus burn to his back from the mishandling of hand grenades.What are the common causes of phosphorus burns?How does phosphorus burns cause injury?What are the clinical features of phosphorus burns?How are phosphorus burns managed?These burns occur mainly in the military and munitions industry. Patients are usually injured by phosphorus-containing bombs and weapons in the field, or during factory production of munitions or fireworks. Barillo et al-4Phosphorus has a high tendency to stick to skin and when in contact, its highly lipid-soluble property can allow deepening of the burn injury into the subcutaneous tissue. Burning white phosphorus produces smoke, composed of mainly phosphorus pentoxide, an irritant to the eyes and mucous membrane of the respiratory tract. Systemic absorption from skin contact or inhalation causes erythrocyte hemolysis and multiple organ dysfunction.2Phosphorus burns are typically severely painful, necrotic, and yellowish in color with a characteristic smell of garlic. They are commonly full-thickness burns, resulting from chemical and thermal insults. Systemic toxicity manifests in 3 phases: (1) In the first 8 hours, patient may experience gastrointestinal symptoms such as abdominal pain, nausea, vomiting, and diarrhoea; (2) From 8 hours to day 3, patient may be asymptomatic; (3) From day 4 to day 8, multiorgan failure and central nervous system dysfunction may result in death. Clinicians should also be wary of predictable complications such as hypocalcemia, hyperphosphatemia, and cardiac arrhythmia.There are no good-quality studies to recommend the optimal management strategy for phosphorus burns. However, there is consensus that thorough irrigation of the wound with saline or water and complete removal of phosphorus particles by debridement are pivotal. Identification of phosphorus particles is facilitated by use of either copper sulphate or ultraviolet light. Copper sulphate converts phosphorus to a black film of cupric phosphide, making small or embedded particles easily visible. However, its use is associated with renal failure and poor outcomes according to a recent Cochrane Review.


Mitochondrial dysfunction has been implicated in the development of insulin resistance (IR); however, a large variety of association and intervention studies as well as genetic manipulations in rodents have reported contrasting results. Indeed, even 39 years after the first publication describing a relationship between IR and diminished mitochondrial function, it is still unclear whether a direct relationship exists, and more importantly if changes in mitochondrial capacity are a cause or consequence of IR. This review will take a journey through the past and summarise the debate about the occurrence of mitochondrial dysfunction and its possible role in causing decreased insulin action in obesity and type 2 diabetes. Evidence is presented from studies in various human populations, as well as rodents with genetic manipulations of pathways known to affect mitochondrial function and insulin action. Finally, we have discussed whether mitochondria are a potential target for the treatment of IR. Mitochondria, originating from bacterial precursor cells that were able to generate energy, provide a platform for the generation of ATP, the energy currency of the cell. As ATP is essential for many cellular processes, mitochondrial function  plays an important role in metabolic health and cellular fate. Mitochondrial function can be defined in a number of different ways, but for the purpose of this review we have largely focussed on the role of mitochondria in metabolic processes including oxidative phosphorylation and substrate oxidation  As well as defining mitochondrial dysfunction, it is important to give an overview about the possible mechanisms by which impairments in mitochondrial oxidative metabolism  could affect insulin sensitivity. Mitochondrial dysfunction can result from a decrease in mitochondrial biogenesis, reduced mitochondrial content and/or a decrease in the protein content and activity of oxidative proteins ‘per unit of mitochondria’ (such as a decrease in the complexes of the electron transport chain (ETC)) . All sucAn additional potential mechanism linking mitochondrial dysfunction to IR is that decreases in substrate oxidation affect electron flow through the ETC, causing electron leakage towards oxygen and the formation of superoxide. Superoxide and other ROS damage various mitochondrial and cellular components , and potentially results in either mitophagy  or, under high stress levels, apoptosis B. Removaet al. 2 oxidoreductase activity and reduced mitochondrial size when compared with lean control subjects. One year later, two major microarray studies carried out in muscle showed mitochondrial biogenesis and oxidative phosphorylation pathways to be downregulated in T2D patients and non-diabetic individuals with a family history (FH+) of T2D when compared with healthy controls Mitochondrial dysfunction was first described in the context of glucose intolerance ∼40 years ago Although several human (as above) as well as rodent studies Besides evidence for scenario 1  and scenario 2 , several research groups, including ours, have shown a compensatory increase in mitochondrial oxidative capacity with increased lipid supply Collectively, the three possible scenarios described above  suggest that mitochondrial dysfunction is not a requisite feature of IR in all circumstances and the presence of mitochondrial dysfunction is dependent on its definition, the population studied, the model system examined (e.g. human vs rodent models) and the methodological approach (e.g. association vs intervention studies).TFAM, PGC1 and acetyl-CoA carboxylase 2 (ACC2), and discuss their effects on mitochondrial function and insulin sensitivity.As association studies of IR and mitochondrial function, as commonly reported for humans, are unable to delineate whether changes in mitochondrial function are a cause or consequence of IR, various groups have relied on genetic manipulations and interventional approaches to define the cause-or-consequence question of whether changes in mitochondrial function have effects on tissue and whole-body insulin sensitivity. Various transgenic approaches have been taken to try to answer this question. As it is not possible to cover all of these studies, we will give examples of alterations in gene expression of three key metabolic regulators, TFAM, whose expression is regulated by nuclear respirator factor 1 (Nrf1) Tfam-knockout mice exhibit abnormally appearing mitochondria in their muscles as well as progressively deteriorating respiratory chain function Tfam-knockout mice and their results suggested that mitochondrial dysfunction in skeletal muscle is not a primary etiological event in the development of IR, as knockout mice exhibited improved glucose clearance during a glucose tolerance test and increased glucose uptake into muscle (likely due to increased expression of the glucose transporters GLUT1 and GLUT4 and increased AMP-activated protein kinase (AMPK) activity) Tfam-knockout mice, and their results are in line with the previous findings of Wredenberg et al. Tfam-knockout mice exhibited decreased mtDNA copy number and diminished protein content of mitochondrially encoded proteins in brown and white adipose tissue, but displayed greater oxygen consumption due to higher uncoupling. Increased mitochondrial oxygen consumption resulted in elevated whole-body energy expenditure, which is a possible mechanism for these mice being protected against diet-induced obesity, glucose intolerance and lipid accumulation in their livers TFAM indicate that experimentally inducing abnormalities in mitochondrial function does not necessarily lead to deterioration of glucose homeostasis and insulin sensitivity, highlighting a dissociation between IR and mitochondrial dysfunction.Mitochondrial biogenesis is a complex process, involving a coordinated regulation of mitochondrial and nuclear genomes. An important protein controlling the transcription of mitochondrial proteins is the nuclear-encoded transcription factor TFAM demonstrated a clear dissociation between mitochondrial function and insulin sensitivity, alterations in gene expression of members of the PGC1 network have shown that metabolic downstream effects are variable, with complex compensatory interactions making interpretation of data difficult. PGC1α is a master-regulator of mitochondrial biogenesis, interacting with a large complement of transcription factors and nuclear hormone receptors that are associated with mitochondrial function  Pgc1α-knockout mice are viable; however they exhibit an abnormal energy metabolic phenotype. With age, Pgc1α-KO mice show an increase in whole-body as well as hepatic fat deposition, which is accompanied by a decrease in liver and muscle mitochondrial oxidative capacity. Surprisingly, KO mice were less susceptible to diet-induced IR than WT controls PGC1α also leads to increased fat mass; however in contrast, glucose tolerance was impaired in old mice (24 months), whereas it was unaltered in young (3 months) KO mice PGC1α knockout, other studies have shown that also muscle-specific PGC1α overexpression causes IR and glucose intolerance, potentially due to increased fatty acid delivery into muscle and decreased GLUT4 gene expression PGC1α and PGC1β has shown that elevated PGC protein levels improve diet-induced IR in muscle Although the knockout of et al. Acc2-knockout in mice resulted in increased whole-body fatty acid oxidation. Interestingly, despite a significant increase in lipid oxidation, Acc2-knockout mice were not protected from high-fat diet-induced weight gain, glucose intolerance and deterioration of skeletal muscle glucose disposal, indicating that simply accelerating mitochondrial fatty acid oxidation alone does not prevent from a deterioration of insulin sensitivity et al. Acc2 in mouse skeletal muscle, as well as inactivation in the germline had no effects on body weight, food intake, body composition or glucose homeostasis as compared with controls on chow or high-fat diet. These recent studies of unchanged skeletal muscle and whole-body insulin sensitivity in Acc2-knockout mice contrast with earlier findings by Abu-Elheiga et al. ACC2 deletion leads to increased fat oxidation, reduced body weight and protection against diet-induced obesity and IR Acc2-knockout mice could potentially be due to multiple experimental differences , but highlight that seemingly identical genetic manipulations can give contrasting results, complicating advances in our understanding on the importance of mitochondrial function in the development of IR.As lipid oversupply is a major factor involved in the development of IR, a prevailing view was that increasing mitochondrial fatty acid oxidation would improve insulin sensitivity. Mitochondrial fatty acid oxidation is, in part, regulated by AMPK, which phosphorylates and inactivates the mitochondrial enzyme ACC2, leading to reduced malonyl-CoA levels, elevated CPT1 activity and increased entry of long-chain fatty acids into mitochondria for oxidation In the section above, we have noted a number of examples emphasising that due to variable adaptations in different mouse lines, more direct genetic manipulation of mitochondrial function has still been unable to clearly define the impact of altering mitochondrial capacity on tissue and whole-body insulin sensitivity. However, since many insulin-resistant individuals do show mitochondrial defects, it is possible that the mitochondrial defects could be a consequence of reduced insulin action, which is discussed below.Insulin is an anabolic hormone that is known to play a major role in the regulation of metabolic pathways and protein synthesis in many tissues. A number of groups have examined the effect of insulin on mitochondrial function (largely via insulin infusions) and have also attempted to selectively alter glucose tolerance and insulin sensitivity in a previously ‘metabolically healthy’ environment to determine whether these changes affect mitochondrial function. The methodological approach taken for the latter has been to manipulate the components of the insulin signalling cascade to determine whether alterations directly affect insulin sensitivity (primary effect) and if changes in insulin sensitivity could potentially lead to alterations in mitochondrial capacity and function (secondary effect).et al. Insulin is known to affect various metabolic pathways in lipid and glucose metabolism, by either affecting gene expression or the phosphorylation of metabolic proteins With regards to direct genetic manipulation of insulin signalling components, the best characterised models have manipulated insulin receptor substrate (IRS) proteins. In skeletal muscle, selective disruption of IRS1 or IRS2 had only minor effects on insulin sensitivity, whereas simultaneous deletion of both members of the insulin signalling cascade had dramatic effects on insulin signalling and glucose tolerance IRS1 and IRS2et al. IRS1 or IRS2 in liver had only minor effects on insulin signalling, whereas disruption of both isoforms led to impaired glucose tolerance and insulin sensitivity In addition to the findings in skeletal muscle, similar mitochondrial impairments were also observed in hepatocytes lacking both IRS1/2 deletion are likely mediated through the IRS-PI3K-Foxo pathway, as additional deletion of Foxo1 (triple knockout) was able to rescue the metabolic phenotype These results in muscle and liver suggest that direct genetic induction of IR has major effects on mitochondrial capacity, and that IR itself is able to lead to the development of mitochondrial dysfunction. Mitochondrial effects of These studies of genetic manipulation of components of the insulin-signaling cascade, as well as direct insulin infusion or insulin deprivation, raise the possibility that mitochondrial defects observed in some insulin-resistant subjects could be a consequence of the IR itself.Ectopic lipid accumulation is commonly observed in obesity and T2D and is one of the earliest changes observed during the development of IR Mitochondria are an important source of superoxide generation in the cell, with complexes I and III of the ETC having the greatest capacity for superoxide production Several studies from the 1990s proposed a link between oxidative stress and the development of IR. Lipid infusions in humans increased plasma thiobarbituric acid reactive substance (TBARS) levels and simultaneously inhibited insulin-stimulated whole-body glucose disposal et al. 2O2-emitting potential of mitochondria without any changes in oxidative capacity. Attenuating mitochondrial H2O2 emission, by treating rats with a mitochondrial-targeted antioxidant or via overexpression of catalase in mouse muscle mitochondria, completely preserved glucose tolerance and insulin sensitivity in vitro studies 2O2 as a primary mechanism in the development of IR.More direct evidence connecting IR and mitochondrial ROS generation came from a recent study by Anderson The studies noted above support a role for oxidative stress in the development of IR, and there have been a number of antioxidant supplementation trials which have reported that decreasing oxidative stress improves insulin sensitivity and glucose tolerance in various human populations These disparate findings suggest that depending on the experimental conditions, oxidative stress may be one of a number of factors contributing to IR. However, in circumstances where elevated mitochondrial ROS production is present during the development of IR, it is likely tightly coupled with autophagic removal of damaged mitochondria, a process termed mitophagy.Upon increased cellular and mitochondrial stress, mitochondria are able to activate both cell death pathways and mitophagy, with the two opposing forces in the cell, demonstrating a tight balance between life and death  (Mfn1/2), both located on the outer mitochondrial membrane, and optic atrophy1 (Opa1), localised on the inner membrane of mitochondria Fis1, located throughout the outer membrane, and the dynamin-related protein 1 (DRP1). DRP1, lacking a mitochondrial targeting sequence, is usually located in the cytosol, but gets recruited to the mitochondrial outer membrane by Fis1 during the initiation of the fission process MFN2 expression  histological staining pattern and no indication of respiratory deficiency), suggesting possible compensatory interactions between both isoforms Mfn2 levels show a positive correlation with insulin sensitivity in morbidly obese subjects and after bariatric surgery To maintain a metabolically healthy environment, removal of damaged mitochondria through mitophagy requires cells to be able to adapt quickly through changes in mitochondrial fusion and fission pathways. Mitochondria are dynamic organelles able to constantly undergo fusion and fission events to maintain their function As described in the preceding sections, there is substantial controversy on the exact role of mitochondria in the generation of IR. However, because of their role as the key site for substrate oxidation and ATP generation, targeting mitochondrial energy metabolism may has some benefit for metabolic disorders such as obesity and T2D. Below we discuss the evidence for various approaches  aimed at improving aspects of mitochondrial function for potential therapeutic purposes. We also highlight the intriguing work showing that under certain circumstances, mild inhibition of mitochondrial function can also result in beneficial metabolic effects.et al. et al. et al. Exercise is known to have major impact on both mitochondrial function and insulin sensitivity in skeletal muscle. A 12-week exercise intervention programme in T2D patients significantly increased muscle mitochondrial respiration and mitochondrial content +-dependent deacetylase sirtuin 1 (SIRT1) is suggested to be the principal modulator of the downstream pathways responsible for the beneficial metabolic effects of calorie restriction In addition to exercise, caloric restriction is an effective treatment improving obesity and IR. In obese humans, caloric restriction and weight loss were shown to improve insulin action The coupling of mitochondria describes the molar ratio of the yield of ATP (per oxygen consumed), also referred to as the P/O ratio, while mitochondrial uncoupling is defined as futile cycling of protons across the mitochondrial inner membrane, without coupling to ATP generation et al. Pharmacological agents, such as 2,4-dintirophenol (DNP), can induce mitochondrial uncoupling by transporting protons across the mitochondrial inner membrane into the matrix. DNP was successfully used as an anti-obesity agent in the 1930s, suggesting that increased energy expenditure through mitochondrial uncoupling could have therapeutic potential 2, with the entry of electrons into the ETC via complex II allowing substrate metabolism to continue despite a transient block at complex I. In addition, a decrease in ROS generation could be potentially mediated via reduced electron backflow and superoxide production from the ubiquinone reduction site of complex I with FADH2 (generated during each β oxidation cycle) as the electron donor.Asian countries have a long history of using natural compounds for the treatment of metabolic disease. One of the compounds is Berberine, a natural plant alkaloid that was shown to improve insulin sensitivity in rodents and humans in vivo, not just in the typical fasting state, but also under a variety of other free-living conditions . Furthermore, increasing evidence of multiple post-translational modifications in mitochondria highlights that future studies in isolated mitochondria or tissue preparations must be performed in the presence of appropriate inhibitors to maintain the mitochondria in a similar state that observed in vivo. Studies such as these may help to more clearly define the extent to which mitochondrial dysfunction contributes to the development of IR, and also determine how effective targeting mitochondria may be for the treatment of IR.During the last 20 years, many studies have reported changes in mitochondrial function in various models of metabolic disease. Interestingly, these changes have been of opposing character, with some studies reporting a decrease in mitochondrial function , while others have reported either no change or an increase in mitochondrial oxidative metabolism in obese and insulin resistant humans and rodents. Similarly, studies using genetically manipulated mice to alter mitochondrial function have also produced confounding results. Collectively, these studies highlight that the relationship between mitochondria and insulin action is highly complex and there is still much to learn in this area. Some potential approaches for closing these gaps in knowledge in future studies include developing methods (e.g. with stable isotope tracers) to assess dynamic changes in mitochondrial function