Agents that augment cerebral blood flow (CBF) could be potential treatments forvascular cognitive impairment. Phosphodiesterase-5 inhibitors are vasodilatingdrugs established in the treatment of erectile dysfunction (ED) and pulmonaryhypertension. We reviewed published data on the effects of phosphodiesterase-5inhibitors on CBF in adult humans. A systematic review according to PRISMAguidelines was performed. Embase, Medline and Cochrane Library Trials databaseswere searched. Sixteen studies with 353 participants in total were retrieved.Studies included healthy volunteers and patients with migraine, ED, type 2diabetes, stroke, pulmonary hypertension, Becker muscular dystrophy andsubarachnoid haemorrhage. Most studies used middle cerebral artery flow velocityto estimate CBF. Few studies employed direct measurements of tissue perfusion.Resting CBF velocity was unaffected by phosphodiesterase-5 inhibitors, butcerebrovascular regulation was improved in ED, pulmonary hypertension, diabetes,Becker's and a group of healthy volunteers. This evidence suggests thatphosphodiesterase-5 inhibitors improve responsiveness of the cerebralvasculature, particularly in disease states associated with an impairedendothelial dilatory response. This supports the potential therapeutic use ofphosphodiesterase-5 inhibitors in vascular cognitive impairment where CBF isreduced. Further studies with better resolution of deep CBF are warranted. Thereview is registered on the PROSPERO database (registration numberCRD42016029668). This issue is complex to resolve becausestandard magnetic resonance imaging (MRI) sequences may not be sufficientlysensitive to detect the earliest pathological changes in SVD.1315 However, reduced CBF may occur prior to the onset of clinical symptomsof Alzheimer's disease (AD),16 and is the earliest observed neurological abnormality in mouse models of AD.17 The mechanisms by which reduced CBF might accelerate SVD pathogenesis areunclear, but reduced protein synthesis is a well-described effect of mild degrees ofcerebral ischaemia.18Cognition declines over time as SVD advances.17 This process can be termed cerebrovascular regulation (CVR). Impaired CVR maybe associated with vascular risk factors and vascular disease in general.20 However,impairment of endothelium-dependent CVR is postulated to be a specific pathologicalfeature of SVD.2124 Although data are limitedthere is some evidence that impaired CVR correlates with the burden of white matterhyperintensities, a key radiological marker of SVD.25 Similarly, haemodynamic pulsatility, a measure of vessel stiffening, has alsobeen shown to strongly correlate with the degree of leukoaraiosis.26 Improving CBF and CVR by augmenting endothelial-myocyte signalling (and thusimproving function of the neurovascular unit) therefore has potential as bothsymptomatic and disease-modifying treatment for SVD.8CBF regulation is dependent on intact endothelial and myocyte function in smallpenetrating arteries, which facilitates increased regional blood flow in response todemand, such as during cognitive activity.27 In PH PDE5 is more active and expressed more abundantly in the pulmonaryvasculature.29 PDE5i reduce this heightened activity (sildenafil increasingcGMP levels 5- to 10-fold) leading to vasodilation. In PH there appear to be furtherdownstream effects, PDE5i leading to reduced DNA synthesis in myocytes and reducedsmooth muscle proliferation.29Phosphodiesterase-5 (PDE5) inhibitors (PDE5i) are used in the treatment of pulmonaryhypertension (PH) and erectile dysfunction (ED). Inhibition of PDE5 reducesbreakdown of cyclic guanosine monophosphate (cGMP), leading to vascular smoothmuscle relaxation in small blood vessels, as for example in the treatment of EDwhere PDE5i delay de-tumescence.3033 Western blot detects PDE5 inboth meningeal and larger cerebral arteries of experimental rodents and humanparticipants35 and immunohistochemical labelling shows that PDE5 is present insmooth muscle cells of small arterial vessels.36 PDE5 activity in SVD has not been reported. This raises the question whetherPDE5i could augment vasodilation of cerebral blood vessels, and hence increase CBFand/or restore CVR in SVD.PDE5 mRNA and protein are expressed in brain tissue of humans and experimentalanimals.The aim of this review is to synthesise published data from human studies on theeffects of PDE5i on CBF and CVR in adults.Protocol: PRISMA guidelines were followed (please see the PRISMA checklist onlineSupplement).Eligibility criteria and study selection: randomized clinical trials andobservational studies investigating the effects of PDE5i on CBF in adult humans wereconsidered for inclusion. Abstracts were considered if data were presented. Allimaging modalities for directly or indirectly measuring CBF and CVR were included.Only papers available in English were included. Publications were excluded if theyhad no adult human data, did not use selective PDE5i, or had no data on measurementsof CBF. Case reports presenting data from single subjects were excluded. Forexcluded studies please see Supplement Table 1.Information sources: (1) Embase, Medline and the Cochrane Library database searcheswere performed initially up to November 2016, and then repeated to check for newpublications on 4 August 2017. Results were reviewed independently by MMHP and JDI.(2) Reference lists of all included studies, as well as of relevant non-eligiblestudies were checked.Search strategies can be found in the online Supplement SII.Data extracted from individual studies were: participant characteristics, presence orabsence of a control group, intervention ,assessment modalities and outcomes.Risk of bias in individual studies was assessed using the Cochrane bias assessmenttool.A qualitative narrative synthesis was performed because the very heterogeneousstudies and data were unsuitable for meta-analysis.n = 103 participants).3742 One was a randomisednon-blinded study (n = 30), the two participant groups receivingtwo different medication protocols.43 Two studies with a total of 68 participants were controlled but notrandomised or blinded.45 Seven observational studies with a total of 152 participantswere included.4652 Details of risk of bias aredisplayed in Sixteen studies with a total of 353 participants were included in the review  reported a clinical improvement in one patient with resolution ofpronator drift after administration of PDE5i.A summary of the outcome measures used in the various studies is shown in VMCA) in one study,37 and an average of maximal velocities in the other,39 both determined by transcranial doppler imaging (TCD). Neither trialfound changes in resting VMCA. In one of these studies39 single-photon emission computed tomography (SPECT) imaging with Xenon 133inhalation was also used to assess regional CBF at rest, with no differencebetween sildenafil and placebo. In a later study using functional magneticresonance imaging (fMRI), sildenafil did not affect changes in CVR triggered byhypercapnia and visual stimuli.47Four studies investigated the effect of sildenafil on CBF in healthy individuals.In two, sildenafil was compared to placebo in a double blind crossover design.The measure of CBF was mean blood flow velocity in the middle cerebral artery was reduced in both the hypercapnia andhyperventilation (i.e. hypocapnia) conditions.52In contrast, a study with 14 healthy male volunteers found that 1 h aftersildenafil (100 mg) CVR was improved, in that 40 investigated the effect of sildenafil on restingVMCA as well as resting global and regional CBF(using SPECT) in 12 migraineurs. Radial and temporal artery diameters were alsomeasured. Ten subjects experienced a migrainous headache after sildenafil, andonly two subjects after placebo. There were no changes in global or regionalCBF, VMCA, or in radial or temporal arterydiameter.A double blind crossover trial41VMCA and an Acute Mountain Sickness (AMS) score weremeasured at an altitude of 490 m and approximately 20 h after a two-day ascentto 4559 m. The cerebral autoregulation index (ARI) was used to assess CVR. ARIwas calculated from the rate of restoration of VMCAfollowing an acute spell of hypotension induced by the sudden release ofbilateral thigh cuffs inflated to 30 mmHg above systolic blood pressure for 3 min.53 Neither tadalafil nor dexamethasone had a significant effect onVMCA or ARI. AMS score improved withdexamethasone and tended to be better with tadalafil than placebo.In a double blind trial 35 healthy individuals were randomly allocated totadalafil 10 mg twice daily, dexamethasone 8 mg twice daily or placebo.n = 10) sildenafil 50 mg was given oneand three days after rapid ascent to 3480 m.46 Measurements were taken prior to dosing and at 1 and 2 h post-dosing oneach day. On day one, systemic oxygen saturation (SaO2) and cerebraloxygenation increased after sildenafil, with unchangedVMCA and end-tidal partial pressure of carbondioxide (pCO2).In an uncontrolled study  were measured 50 min after a singledose of sildenafil (50 mg). FVD was derived from a comparison between breathholding  and hyperventilation(leading to decreased pCO2 and vasoconstriction);54 . No difference was found in restingVMCA 1 h post-dosing with sildenafil compared toplacebo. However, sildenafil significantly improved cerebrovascularreactivity.One double-blind randomised controlled trial compared sildenafil (50 mg) with placebo48 Both used pre- andpost-dose SPECT imaging to assess regional CBF. The initial study48 enrolled 25 male patients with ED, of whom 12 had a history of ischaemicstroke 1–5 years prior to study participation. Among the other 13 participants,eight had non-territorial focal areas of diminished CBF on baseline SPECT, andfive had diffuse CBF abnormalities suggestive of SVD. The extent and exactlocation of white matter intensity lesions were not reported. One hour afteroral administration of sildenafil (50 mg) the post-stroke group showed moreareas with relatively diminished perfusion, and fewer areas of relativelyincreased perfusion, compared to non-stroke patients, although there wasvariation between subjects within the group. It was not possible to identify aclear pattern from the Brodman areas that were listed as showing a significantdifference .Two studies investigated the effects of PDE5i on CBF in patients withcerebrovascular disease and ED.43 all participants had ED and a history of large vessel ischaemic or smallvessel ischaemic stroke  that had occurred 3–36 months prior to the study. Lesion volumeswere not reported. Participants were randomised to either single dose tadalafil(20 mg) or daily dosing of tadalafil (5 mg) for seven days. SPECT was performedat baseline and 6 h after the final (or single) dose.In the second studyReduced relative CBF was found in the stroke-affected hemisphere as a wholeversus the unaffected hemisphere after tadalafil, in both treatment arms,particularly in the zones bordering the infarct. However, smaller cortical andsubcortical areas showed mixed effects (with areas of increased and decreasedflow on either side), with no clear region or side-specific pattern. Nosignificant difference was found between the two stroke groups  or the two dosing regimens.4951 One study49 enrolled 72 patients with vasospasm refractory to 24 h of hypertension,hypervolaemia and haemodilution (HHH) therapy. All patients had had theiraneurysms surgically secured and had received nimodipine. Followingadministration of 100–150 mg of intravenous sildenafil every 4 h (for 2–7 days)eight showed sustained (>48 h) reduction in VMCA(>40 cm/s), and five had intermittent reductions.Three uncontrolled studies investigated the effects of sildenafil during theacute phase of SAH.50 12 patients with angiogram proven vasospasm received intravenoussildenafil (10–30 mg). Angiography was repeated 30 min after infusion. Eightpatients had an improvement in vasospasm.In another study15O2-positron emission tomography (PET)imaging was performed pre- and post-intravenous sildenafil in six patients withdigital subtraction angiography proven SAH.51 No changes in regional or global CBF were found after sildenafil.In a further study None of these studies had a control group or control condition.45 Flow velocity of the posterior cerebral artery(VPCA) was recorded at baseline (where it wassignificantly lower in those with PH versus controls), and in response to avisual stimulation paradigm. Pulmonary arterial pressure and systemic vascularresistance were also assessed. The 22 healthy control participants underwent thevisual stimulation and VPCA measurements but werenot given the study medication and did not have right heart catheterisation. Insubjects with PH both agents reduced pulmonary and vascular resistanceparameters. The attenuation parameter  and the time rate parameter  were also assessed. Sildenafiladministration was associated with normalization of both of these in PH. Incontrast, iloprost slightly worsened the time rate parameter.In 11 adults with severe PH, inhaled iloprost and 50 mg oral sildenafil wereadministered consecutively (with a washout period).42 A randomised double blind cross-over trial of 12 participants with BMDcompared the effect of sildenafil with placebo in a four-week treatmentblock.In BMD the dystrophin protein is absent or truncated leading to loss of neuronalnitric oxide synthase (nNOS) and nitric oxide (NO) production, causingfunctional ischaemia in skeletal muscle, and possibly also in cerebral vascular myocytes.CVR in response to sensory stimulation was examined using blood oxygenation-leveldependent (BOLD) functional MRI. Compared to placebo, sildenafil significantlyincreased BOLD signal in the somatosensory cortex in response to median nervestimulation. Increased BOLD response was also observed in the visual cortexfollowing a flickering checkerboard stimulus.2 reactivity, measured using MR phase contrastmapping and MR angiography, were unaffected by sildenafil.MCA circumference and CO3751 with the possibleexception of SAH.50 In contrast, CVR in states of acute challenge, such ashyperventilation or stimulatory tasks, was improved by PDE5i. This was observedin clinical conditions associated with impaired endothelial vasodilatoryresponses,45 and also in one study of healthy volunteers.52In the studies retrieved resting CBF was not affected by PDE5i in health ordisease states56 Contractile messengersinclude the sympathetic neurotransmitters norepinephrine and neuropeptide Y.Sympathetic stimulation via beta-adrenoceptors augments L-type calcium channelactivity. Relaxant mediators include the parasympathetic transmitteracetylcholine, vasoactive intestinal peptide, calcitonin gene-related peptideand substance P. Many messengers converge on a key signalling pathway:endothelial derived NO.56 NO activates soluble guanylate cyclase in myocytes, whichconverts guanosine triphosphate to cGMP. As cGMP concentration increases, smoothmuscle tone is reduced and the vessel dilates.5557Vascular smooth muscle tone regulation is complex, with inputs from neurons,glia, interneurons and perivascular nerves.29 and ED,27 and is augmented in both conditions by PDE5i treatment. A similarmechanism may also apply to the cerebral circulation. This is supported by thefact that endothelial cells have an important role in CBF regulation.25 Vesselwall morphology and function are affected in SVD.59 The PDE5i-mediatedimprovement in CVR noted here, in individuals with diseases affecting vascularfunction48 may reflect augmented responsiveness of the endothelium andneurovascular unit to local metabolic or neurogenic stimuli.Altered NO-cGMP signalling has been shown to be a feature of PH42 supports this theory, describing ‘functional ischaemia’ due to reducedfunction of nNOS, rather than endothelial NOS (eNOS), and showing augmentedresponses to stimuli following treatment with sildenafil (see below).42 Although a different subtype of NOS is implicated, these findings supporta role for the NO-cGMP signalling pathway in CVR.The study investigating BMD60 the vasodilatory effects of PDE5i  are well-described.6164 Furthermore, PDE5iattenuate vascular remodelling and right ventricular hypertrophy, as well asimproving functional status in PH.66In PH, where endothelial PDE5 is expressed more abundantly than in healthylung,52 but not in a study of healthy females using fMRI,47 or in the healthy control group in another study.44 Differences in imaging modalities and different sildenafil doses  may explain thesedivergent findings. The effectiveness of the hypercapnic stimulus may havevaried between studies; only Jahshan et al.53 confirmed a rise in pCO2 and were also the only authors toreport an effect of PDE5i on CVR in healthy volunteers. Furthermore, all threeof these studies had small sample sizes.It is interesting that a similar response was found in one study in healthy malesassessed by TCD in response to hypercapnia and hypocapnia,26 Of the studies here, only one investigated haemodynamic pulsatility,using PCA attenuation parameter  and time rate parameter .45 Both measures were improved by sildenafil in participants with PH. Thisraises the question whether PDE5i might delay the progression of SVD bymitigating the deleterious effects of raised large artery pulsatility on thesmall vessels.SVD is also associated with proximal vessel stiffness and pulsatility; increasedMCA pulsatility has been proposed to damage the microvasculature by transmittinghigher pressure differences between systole and diastole to the cerebral small arteries.The lack of change in basal CBF following PDE5i, in the papers retrieved here,may reflect the absence of a stimulus. Another explanation is that thetechniques used were insufficiently sensitive to detect modest changes inresting CBF in relevant brain regions .VMCA3752) or posterior cerebralartery (VPCA45). Although TCD is non-invasive, affordable, portable and readilyrepeatable, and gives ‘real time’ readings of flow velocity, it has importantlimitations. TCD assesses flow velocity in one large vessel only, and thus onlyindirectly the overall blood supply to the brain as a whole. Importantly, itdoes not report on the smaller, deep brain vessels implicated in SVD, and thedistribution of flow across these. Further, it is likely incorrect to assumethat constant VMCA implies no change in blood volumedelivered to the brain. Specifically, this inference must assume a constantdiameter of the vessel measured. In contrast, in patients with SAH, decreasedVMCA is interpreted as MCA dilatation reflectinggreater global CBF.50 Even very small changes in vessel diameter imply verysignificant changes in flow at a given velocity as vessel resistance is relatedto vessel radius to the power of four. Although accurate measurement of vesseldiameter would thus be key to determining blood flow, none of the TCD studiesretrieved here directly measured MCA diameter; one study measured radial andtemporal artery diameter (both unchanged), and calculated MCA diameter fromSPECT assessment of CBF and VMCA.40 MCA diameter assessed by magnetic resonance angiography (MRA) in the BMDstudy was unchanged,42 and increased in some participants in one of the SAH studies, where itwas measured by digital subtraction angiography.50 In light of these reports, we believe that assumptions regarding MCAdiameter should be viewed with caution.Among the included studies, 10 used TCD to measure CBF velocity in the middlecerebral artery .42 In two studies using HMPAO SPECT in individuals with prior stroke, arelative reduction in regional CBF in and around the stroke lesion was reported,with increase in some other brain areas.48 The authors ascribe thisto post-stroke dysfunction of cerebral auto-regulation, leading to a ‘stealphenomenon’ whereby healthy brain is able to achieve a greater vasodilatoryresponse at the expense of ischaemic areas.67 However, infarcted areas will not regain blood flow. Around the infarct,there may also be areas of oligaemia, which have only limited supply fromcollateral vessels.6870 Underlyingpathology and mechanisms in large vessel strokes are different to those in SVD,so these findings may not be generalisable. Furthermore, measurements ofreduction in relative flow in areas immediately adjacent to infarct areas mayalso be due to sub-optimal spatial resolution of SPECT, with voxels overlappingthe infarcted area, i.e. partial volume effect.72Measurement of CBF can also be performed using SPECT, PET, fMRI and arterial spinlabelling (ASL) MRI, which allow resolution of regional CBF. The studies usinghexamethylpropyleneamineoxime (HMPAO) or Xenon SPECT.It is not currently known whether changes in CVR reviewed here affect cognitiveperformance, or alter disease progression in SVD.4 An attemptto reduce cognitive decline in SVD through blood pressure control andantiplatelet treatment was negative in a phase III study monitoring participantsfor a mean of three years.73In vascular cognitive impairment there is a lack of consensus regarding diseaseclassification and diagnostic criteria, and the exact relation between pathologyand cognition has not been determined. Patient groups in trials are thus oftenheterogeneous  so that it has beendifficult to identify therapeutic targets and treatments.74 However, studies of PDE5i in animal models of neurodegeneration haveshown promising results. A study using sildenafil found improved memory taskperformance in a mouse model of AD.75 In a similar mouse model tadalafil improved water maze performance andreduced Tau phosphorylation after 12 weeks of treatment.76 Object recognition task performance was also improved by PDE5i in maleWistar rats.77 Acute stroke studies in rodents found improved outcome with PDE5i,apparently due to neurorepair.7881Pre-clinical studies of the effects of PDE5i in cerebrovascular disease arelimited by the lack of a robust animal model of SVD.82 In another study of six healthy participants sildenafil enhancedperformance in a simple reaction time test, but not on other measures (includingshort term memory and divided attention).83Studies of the effects of PDE5i on cognition in humans are limited. A study ofsildenafil in 10 healthy males found enhanced event-related potentials, but nochanges in auditory attention and word recognition.84 and in an individual with spastic quadriplegia, mild motor restorationwas found after administration of sildenafil (the details of this are notfurther specified).85Beneficial neurorestorative and functional effects of sildenafil have beenobserved in two case reports. In a subject with a history of occipital infarcts,visual field defects improved following sildenafil treatment,83 can be sustained with longer-term administration isunclear. However, the case reports of single subjects with disease states(spastic quadriplegia and stroke) reported improvements in symptoms lasting seven85 to over 12 months84 in motor and visual symptoms, respectively.Whether the cognitive effects observed in the two single-dose studies in healthyindividuals3742 Individually these trials hadsmall sample sizes, ranging from six37 to 35 (divided into three treatment and control groups).41 None of the remaining studies were blinded and only a further two had controlgroups.45 Results should thus be interpreted with caution.The review is limited by the relative paucity of research studies and variability indata quality. Of the included studies, only six were double blind randomisedcontrolled trials  with a totalof 103 participants.The participant groups in the studies included here are very heterogeneous with avariety of brain and/or systemic pathologies and hence potentially disparate diseasemechanisms. Protocols of intervention, PDE5i used, route of administration andoutcome measures differed between studies, with a range of techniques used tomeasure CBF.42 the stroke and ED study comparing once only tadalafil with a seven day regimen43 and one of the altitude trials, where PDE5i was given for about four days.46 Nevertheless, none of the interventional studies investigated periods longerthan a number of weeks. One of the altitude studies, however, showed attenuation ofsome of the observed changes on day three versus day one.46 This raises the question whether any short-term effects on CVR observed willbe maintained with longer-term administration.It should also be noted that most of the retrieved studies only assessed acute (oftensingle) dosing of PDE5i. Exceptions to this are the BMD study, where sildenafil wasgiven for four weeks prior to assessment,48 and PH studies,45 where this may be expected (the study in PH included participants ‘using ornot using sildenafil’48). Only three studies explicitly state that subjects were not using47 or were notprevious users50 of PDE5i.Whether or not PDE5i had been used prior to study intervention by participants is notreported, particularly in the ED49), thus females are significantly under-represented, particularly in thestudies investigating disease groups. Due to these factors comparison across studiesis imperfect and findings cannot easily be generalised.Across all studies, of the 353 participants only 53 were female  are warranted to explore effects of PDE5i at the arteriolar level. Futurestudies should also test whether PDE5i mediated vascular changes are correlated withcognitive function, using tools that assess appropriate cognitive features such asattention, information processing speed and executive function, which areparticularly affected in SVD.87Future studies using high resolution, blood-flow specific cross-sectional imagingtechniques as surrogates of deep CBF measurements (e.g. ASL MRI


Cryptorchidism or undescended testis (UDT) is a common congenital abnormality associated with increased risk for developing male infertility and testicular cancer. This study elucidated the effects of endogenous ghrelin or growth hormone secretagogue receptor (GHSR) deletion on mouse reproductive performance and evaluated the ability of ghrelin to prevent testicular damage in a surgical cryptorchid mouse model. Reciprocal matings with heterozygous/homozygous ghrelin and GHSR knockout mice were performed. Litter size and germ cell apoptosis were recorded and testicular histological evaluations were performed. Wild type and GHSR knockout adult mice were subjected to creation of unilateral surgical cryptorchidism that is a model of heat-induced germ cell death. All mice were randomly separated into two groups: treatment with ghrelin or with saline. To assess testicular damage, the following endpoints were evaluated: testis weight, seminiferous tubule diameter, percentage of seminiferous tubules with spermatids and with multinucleated giant cells. Our findings indicated that endogenous ghrelin deletion altered male fertility. Moreover, ghrelin treatment ameliorated the testicular weight changes caused by surgically induced cryptorchidism. Testicular histopathology revealed a significant preservation of spermatogenesis and seminiferous tubule diameter in the ghrelin-treated cryptorchid testes of GHSR KO mice, suggesting that this protective effect of ghrelin was mediated by an unknown mechanism. In conclusion, ghrelin therapy could be useful to suppress testicular damage induced by hyperthermia, and future investigations will focus on the underlying mechanisms by which ghrelin mitigates testicular damage. Cryptorchidism, also known as undescended testis (UDT), is one of the most common abnormalities of male sexual development characterized by failure of the testis to descend into the scrotum where temperature is optimal for spermatogenesis . Higher Ghrelin, a n-octanoylated 28 amino acid peptide, has been identified as the endogenous ligand for a specific G-protein-coupled receptor called growth hormone secretagogue receptor (GHSR) , 13. TwoThe direct action of ghrelin in inhibiting apoptosis has been demonstrated in multiple cell types, including cardiomyocytes , 24, pitThe present study was designed to test whether ghrelin and its functional receptor GHSR1a are key mediators in mouse spermatogenesis to improve the understanding of mouse reproductive biology. We demonstrated that ghrelin knockout (KO) mice have a significant increase in germ cell apoptosis and a significant decrease in litter size for breeding pairs that included ghrelin KO males. While we hypothesized that the GHSR pathway, following ghrelin administration, was involved in preventing surgically-induced testicular damage we found that this protective response was also detected in GHSR KO mice, indicating that the ghrelin induced antioxidant effect may be mediated by a second receptor that has not been identified.ad libitum. Mice were housed in the Brown University Animal Care Facility and kept in a continuous 12 hrs alternating light-dark cycle with controlled temperature (25–28°C) and humidity (30–70%). All investigations were conducted in accordance with The Guide for the Care and Use of Laboratory Animals and were approved by the Brown University Institutional Animal Care and Use Committee (IACUC protocol number: 1412000111).Male wild type C57BL/6 mice (n = 80), 50–55 days old, were purchased from Charles River Laboratories . All mice were allowed free access to Purina Rodent Chow 5010  and water -/-) or ghrelin (ghrelin-/-), were developed at Baylor College of Medicine  [Growth hormone secretagogue receptor knockout (GHSR KO) mice and ghrelin knockout (ghrelin KO) mice, C57BL/6 mice with a deletion of the growth hormone secretagogue receptor  , 36. GHSGHSR knockout transgenic mouse line .ghrelin knockout transgenic mouse line .Females and males of heterozygous (+/-) or homozygous (-/-) ghrelin or/and GHSR knockout mice were paired and the newborn pups were counted for every mating pair. Pups were tail clipped before 21 days of age for genotyping in accordance with Brown’s “Mouse Tail Biopsy Policy” approved by the local ethics committee. Testes of adult mice were collected, weighed and embedded in paraffin for histological analysis.To examine the morphological appearance of seminiferous tubules of both the transgenic mouse lines, testes were collected and fixed overnight in Bouin’s solution, dehydrated in a descending series of ethanol washes and embedded in paraffin. 5 μm sections were stained with hematoxylin and eosin (H&E), cleared with isopropanol and Citrisolv , mounted using Cytoseal-60 (Thermo Fisher Scientific) and examined under a light microscope.Germ cell apoptosis was detected in 5 μm paraffin sections of wild type (WT), ghrelin knockout (ghrelin KO) and GHSR knockout (GHSR KO) mice testes using the Apoptag Peroxidase In Situ Apoptosis Detection Kit  according to the manufacturer’s instructions. Slides were scanned into an Aperio ScanScope CS microscope . TUNEL-positive cells and seminiferous tubule diameter were measured using Leica ImageScope software . TUNEL positive cells were counted in an average of 152 ± 24 , 169 ± 33  and 148 ± 22  seminiferous tubules for each cross section having a ratio major axis/minor axis < 1.5. The data were expressed as the percentage of round seminiferous tubules with more than 3 TUNEL-positive cells.1 mg rat lyophilized acylated ghrelin  was dissolved in 1 ml of sterile saline .A total of 80 male C57BL/6 wild type mice and 20 male GHSR KO, 50–55 days of age and weighing between 19–21 g, underwent a midline laparotomy to establish a surgical unilateral cryptorchid model. They were randomly separated into two groups to receive saline as control group and ghrelin as treatment group. In the control group, C57BL/6 wild type and GHSR KO mice were injected intraperitoneally twice daily with 0.1 mL saline, whereas in the treatment group, animals received intraperitoneal injections of 0.16 mg/kg ghrelin diluted in 0.1 mL saline twice daily for 1 day post-surgery , 4 days post-surgery  or 20 days post-surgery . Mice were euthanized by isoflurane overdose according to IACUC-approved protocols.Surgical procedure for induction of unilateral cryptorchidism was performed. Anesthesia was induced with isoflurane  gas and maintained with 2–3% isoflurane via a nose cone throughout the entire sterile surgical procedure. After anesthesia, the surgical area was shaved and prepared by povidone iodine solution. A midline abdominal incision was made, and the right testis was manipulated into the abdomen and sutured to the abdominal wall by a 6–0 gut absorbable suture . For each animal the right testis was treated as the experimental organ, whereas the left testis remained completely untouched throughout the procedure and acted as a control. After the laparotomy incision was closed into layers with a 5–0 Vicryl suture .After the mice were euthanized at 1, 4 and 20 days post-surgery, cryptorchid and normal testes were collected and weighed. The testes were cut in half. One half was fixed in Bouin’s solution to be embedded in glycol methacrylate  for histological analysis and the other half was snap-frozen in liquid nitrogen for glutathione (GSH) content analysis. The frozen half of the testes were thawed and manually homogenized in cold phosphate-buffered saline pH 7.4 (PBS) and 2mM EDTA (ice cold), 1 ml of PBS/EDTA per 10 mg of tissue. Insoluble material was removed by centrifugation at 4°C, 10 minutes, 14,000 g, and the supernatant was collected and recovered for GSH-Glo Glutathione Assay .Cross-sections from the middle of each testis embedded in glycol methacrylated  were stained with periodic acid Schiff’s reagent and hematoxylin (PASH). Blinded slides were examined on an Olympus BH-2 light microscope  for quality of histology and scanned into an Aperio ScanScope CS microscope . Histological endpoints were measured using Leica ImageScope software. The following endpoints were used to assess testicular damage: testicular weights, seminiferous tubule diameter, percentage of tubules with spermatids, and percentage of tubules with giant cells.Total amount of glutathione (GSH) was evaluated by GSH-Glo Glutathione Assay kit  according to the manufacturer’s instructions. Normal cells possess a well-developed biochemical defense system, comprising free radical scavengers such as glutathione (GSH), vitamin C, vitamin E, glutathione peroxidase (GPx), superoxide dismutase activity (SOD) and tissue catalase activity (CAT) . In addiStatistical analysis was performed using GraphPad Prism software . Multiple comparisons between breeding pairs of ghrelin and GHSR heterozygous (+/-) and knockout (-/-) knockout mice or multiple were obtained using a one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. Multiple comparison between saline- and ghrelin-treated wild type and GHSR KO normal and cryptorchid testis were conducted using a one-way analysis of variance (ANOVA) with Fisher’s LSD test. One-way analysis of variance (ANOVA) with Dunnett’s post-hoc test was performed to compare the experimental groups to the normal wild type samples. Finally, two-way analysis of variance (ANOVA) with Fisher’s LSD test was used to compare the ratio of cryptorchid to scrotal testis between ghrelin- and saline-treated wild type mice at 1, 4 and 20 days post-surgery. All data were presented as mean ± standard error of the mean (SEM). Values were considered to be significant at p < 0.05.The mean litter size of wild type C57BL/6 mice has been previously reported as approximately 7 pups per litter . No diffThe impact of ghrelin or GHSR deletion on reproductive success was assessed by analyzing litter size after mating heterozygous and/or homozygous knockout mice. Four groups of breeding pairs for the ghrelin knockout transgenic mouse line with the following genotypes were housed together for 10 days: female ghrelin +/- and male ghrelin +/- , female ghrelin +/- and male ghrelin -/- , female ghrelin -/- and male ghrelin +/- , female ghrelin -/- and male ghrelin -/- . No litters were born from 3 female ghrelin +/- X male ghrelin -/- pairs, 4 female ghrelin -/- X male ghrelin -/- pairs, 1 female ghrelin +/- X male ghrelin +/- pair and 1 female ghrelin -/- X male ghrelin +/- pair. Female ghrelin +/- and male ghrelin -/- breeding pairs  and female ghrelin -/- and male ghrelin -/- breeding pairs  showed a significant decrease in the number of pups per litter compared to the female ghrelin +/- and male ghrelin +/- breeding pairs  designed as the control group. No significant differences were detected for female ghrelin -/- and male ghrelin +/- breeding pairs in the number of pups per litter compared to the control group .Four groups of breeding pairs for the GHSR knockout transgenic mouse line with the following genotypes were housed together for 10 days: Female GHSR +/- and male GHSR +/- , female GHSR +/- and GHSR -/- , female GHSR -/- and male GHSR +/- , female GHSR -/- and male GHSR -/- . No litters were born from 2 female GHSR +/- X male GHSR -/- pairs and 2 female GHSR -/- X male GHSR -/- pairs. No significant differences were detected in the number of pups per litter compared to the female GHSR +/- and male GHSR +/- breeding pairs designed as control group .The right testis weight of ghrelin KO, GHSR KO and wild type male mice was assessed. The testicular weights were significantly increased  in ghrelin KO mice  compared to the wild-type  and GHSR KO mice . No significant differences in testis weight were detected between GHSR KO mice and wild-type mice .Histological cross sections of wild type , ghrelinTestes of the mature male mice surgically confined into the abdominal cavity showed a significant increase in the testicular weight at 1 day post-surgery  0.095 g 0.066 g 0.024 g. At 4 daSevere time-dependent disorganization of the seminiferous tubules was observed in cryptorchid testes. Histopathological examinations showed deterioration in germinal epithelium and seminiferous tubules. At 20 days post-surgery all germinal elements of the tubules were lost with a single layer of Sertoli cells next to the basement membrane of the tubules (data not shown). Examination of Periodic Acid Schiff and Hematoxylin-stained sections of cryptorchid testes from wild-type and GHSR KO mice at 20 days post-surgery was performed. Saline-treated cryptorchid wild type and GHSR KO mice showed degeneration of seminiferous tubules with an accumulation of multinucleated giant cells at 20 days after surgery . MultinuChanges in glutathione (GSH) content levels in cryptorchid testes of wild type and GHSR KO mice compared to normal testes at 20 days after surgery were evaluated. GSH concentration was significantly decreased in saline-treated cryptorchid wild type  and GHSR KO  mice compared to normal testes . Interestingly, ghrelin administration increased GSH concentrations in wild type  and GHSR KO  cryptorchid mice decreasing the gap in the GSH levels between cryptorchid and normal testes .The present study examined the effect of ghrelin on mouse reproductive performance and demonstrated that the protective effects of ghrelin on heat-induced testicular degeneration are not mediated by GHSR-1a receptor. Ghrelin and its functional GH-secretagogue receptor (GHSR) have been detected in rat testes . The tesIn this study, we demonstrated that ghrelin and not GHSR1a deletion in mice affect male but not female fertility. To evaluate the reproductive performance of these transgenic mouse lines, female and male heterozygous and homozygous ghrelin knockout mice were paired and the number of pups was measured. Breeding pairs that included ghrelin KO male mice showed a significant decrease in the number of pups ; howeverProgrammed germ cell death occurs spontaneously during spermatogenesis at a low rate. Normal loss of testicular germ cells is required to limit the number of cells that may be supported by the Sertoli cells –49. IncrCryptorchidism is a common congenital abnormality characterized by a failure of descent of one  or both  testes increasing the risk for developing male infertility, testicular cancer, germ cell loss and impaired spermatogenesis –56. CongSpermatogenic arrest in experimental cryptorchid mice was associated with the formation of multinuclear giant cells and reduced spermatid number in seminiferous tubules. The present study demonstrated that ghrelin treatment increased the percentage of seminiferous tubules containing spermatids in cryptorchid testes of GHSR KO mice, indicating that this effect was not mediated by GHSR1a receptor but through an unknown pathway. No differences were detected in the seminiferous tubule diameter and in the percentage of seminiferous tubules with multinucleated giant cells in cryptorchid wild type mice between ghrelin and saline groups. On the contrary, ghrelin treatment significantly increased seminiferous tubule diameter in GHSR KO cryptorchid mice testes. The protective effect of ghrelin was enhanced in GHSR KO mice in comparison to wild type mice. One possible explanation is that the absence of the GHSR-1a receptor could activate a compensatory effect, increasing the functional impact of ghrelin. Previous findings reported that this protective effect of ghrelin on the testicular germ cells following surgically induced cryptorchidism seems to be mediated by its antioxidant properties . Since aIn conclusion, our data have provided direct evidence that the absence of ghrelin has no effect on female reproductive performance, but does compromise male fertility. In addition, ghrelin ameliorated the adverse consequences of surgically induced cryptorchidism, though the GHSR-1a receptor signaling pathway was not required for this protective effect. Therefore, ghrelin may be a viable therapeutic strategy to accelerate the recovery of testicular germ cells following testicular injury.


With the advent of highly active antiretroviral therapy (HAART) survival rates among patients infected by HIV have increased. However, even though survival has increased HIV-associated neurocognitive disorders (HAND) still persist, suggesting that HAART-drugs may play a role in the neurocognitive impairment observed in HIV-infected patients. Given previous data demonstrating that astrocyte senescence plays a role in neurocognitive disorders such as Alzheimer’s disease (AD), we examined the role of HAART on markers of senescence in primary cultures of human astrocytes (HAs). Our results indicate HAART treatment induces cell cycle arrest, senescence-associated beta-galactosidase, and the cell cycle inhibitor p21. Highly active antiretroviral therapy treatment is also associated with the induction of reactive oxygen species and upregulation of mitochondrial oxygen consumption. These changes in mitochondria correlate with increased glycolysis in HAART drug treated astrocytes. Taken together these results indicate that HAART drugs induce the senescence program in HAs, which is associated with oxidative and metabolic changes that could play a role in the development of HAND. With the advent of highly active antiretroviral therapy (HAART), HIV infection has transitioned from an acute, terminal illness to a chronic but manageable condition . The HIVIn vitro, there has been evidence of HAART drugs inducing ER stress . Cells were cultured until they reached ∼80% confluence before passaging. At each passage, astrocytes were trypsinized, counted, and the cumulative population doubling level (CPDL) was calculated as we have previously described , monoclonal; BD Biosciences, San Jose, CA, United States]; p21 ; phosphorylated (9211) and total p38 (9212) both polyclonal ; phosphorylated (3033) and total p65 (3034) both polyclonal ; Oxphos cocktail of mitochondrial ETC complexes I [ab110242 (20E9DH10C12)], II [ab14714 (21A11AE7)], III [ab14745 (13G12AF12BB11)], IV [ab110258 (12C4F12)], and V [ab14748 (15H4C4)] ; β-actin [A00702 (2D1D10), monoclonal; Genscript, Piscataway, NJ, United States); and β-tubulin [sc-9104 (H-235), polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA, United States). Clone numbers are in bold.Following indicated treatment times, HAs were lysed in radioimmunoprecipitation assay (RIPA) buffer. Western blot analysis was performed under standard conditions using 15 μg of total cell proteins. Membranes were probed for antibodies against p16 [sc-56330  following incubation. All other compounds were washed with PBS. Mitochondrial superoxide levels were assayed by incubating the cells with 5 μM Mito-Sox Red . Mitochondrial membrane potential was detected by incubating cells with 25 nM tetramethylrhodamine (TMRE) . Mitochondrial mass was evaluated by incubating the cells with 100 nM Mito-tracker Green FM . The glucose analog 2-NBDG uptake was detected by incubating the cells with 10 μM of 2-NBDG . For the previous analyses, incubation was performed at 37°C in 5% CO2 for 30 min except 2-NBDG which required 90 min. Cells were collected in 0.25% trypsin-EDTA with complete medium. Cells were analyzed by flow cytometry using a Guava EasyCyte Mini and the Guava Express Plus program . Acquisitions involved 5000 events.Determination of total cellular reactive oxygen species (ROS), mitochondrial ROS, mitochondrial membrane potential, mitochondrial mass, and glucose uptake assessments was made using flow cytometry as previously described . Total cFollowing the end of the treatment period, HAs were incubated with serum-free MCDB105 media. After a 24-h incubation period, media were collected and cells were trypsinized and counted to determine the cell number for normalization. Human Cytokine Array C5  was used to evaluate secreted inflammatory factors in the conditioned media according to the company’s protocol. The intensity of the signal on the array membranes was quantified by densitometry using ImageJ software and normalized to cell number. The HAART drug treated values were then set as relative to control values. Samples that had no change in expression due to levels being undetectable from background were set to 1. Interleukin-6 (IL-6) detection was performed via the Human IL-6 Quantikine ELISA kit  according to the product manual using conditioned media as described above. Absorbance was measured at 450 nm.p-triflouromethoxyphenylhydrazone (FCCP), and rotenone/antimycin A prior to measurement. Oxygen consumption was measured in triplicate before and after consecutive addition of oligomycin, FCCP, and rotenone/antimycin A. Respiration rates and proton leak were assessed as previously described  and the XF Cell Mito Stress Test Kit as previously reported . Cells wescribed  based upMetabolite measurements were performed by Human Metabolome Technologies America Inc.  using their C-Scope analysis. Samples were collected according to their protocol and sent overnight on dry ice, after which capillary electrophoresis mass spectrometry was performed. Quantifications were performed by hierarchical cluster analysis (HCA) and principal component analysis (PCA) by Human Metabolome Technologies America Inc.’s statistical software.t-test when two groups were involved or one-way analysis of variance (ANOVA) followed by Bonferroni correction when three groups were analyzed. Normality was determined using a Shapiro–Wilk Test with the caveat that low sample sizes can reduce the accuracy of normality tests. Means were derived from at least three independent experiments. Error bars on graphs reflect standard deviation (SD). Statistical significance was considered at p < 0.05.Data were either compared using a two-tailed Student’s Figure  which paralleled an increase in SA β-gal activity Figures . Protein levels of the senescence marker p21 but not p16 was significantly increased in the ABC–3TC–RTV combination, suggesting that the pathway may be p21-dependent Figure . However, the ABC–3TC combination did not show a significant increase in either p16 or p21. In order to rule out the induction of apoptosis, cell viability was measured 1 and 4 days after treatment. No reduction in cell viability was observed compared to the vehicle Figure . These results suggest that the ABC–3TC and ABC–3TC–RTV induced reduction in HA growth is the result of induction of the senescence program.HIV-infected patients do not take individual antiretroviral drugs but rather they are put on a regimen that includes several different drug classes. We therefore evaluated whether a clinically relevant combination of HAART drugs could affect physiology of HAs. Cells were chronically treated with the either the nucleotide reverse transcriptase inhibitors (NRTIs) ABC and 3TC alone or in combination with the protease inhibitor RTV. Determination of the effects on cell proliferation indicates that after 1 week of treatment these drugs induce a reduction in the cell number compared to vehicle control , which has been shown to mechanistically induce senescence , a product produced in the first step of glycolysis trends upward but does not reach statistical significance with HAART drug treatment in HAs Figure . On the other hand, pyruvate, the last product of glycolysis, does show a statistically significant increase Figure . Correspondingly, we observed an increase in the production of lactate Figure , indicating that anaerobic glycolysis is enhanced in response to HAART. This increase in lactate production may explain the increased acidification determined by the Seahorse bioanalyzer Figure . Overall, these results indicate that HAART drug treatment induces an increase in glucose metabolism in HAs.Highly active antiretroviral therapy drug treatment severely affected astrocyte mitochondrial respiration accompanied by an increased medium acidification, suggesting an altered lactate production as a consequence of enhanced glycolysis. These results are intriguing because senescent fibroblasts were shown to have profound metabolic changes including increased glycolysis . We therThe HIV-infected population is growing older and with this increased age, a larger risk for age-associated disease . NeuroloWe evaluated the impact of widely used NRTIs, NNRTIs, and PIs on primary HAs. These drugs induced various aspects of the senescence program including decreased cellular proliferation, increased SA β-gal and expression of the cell cycle inhibitor p21. While astrocyte senescence has not been explicitly studied in HIV, there is evidence of astrocytes and other glial cells expressing cell cycle inhibitors common to senescence in HIV-infected patients . In concin vivo since inflammation may contribute to age-related decline in organ function  treatments were done using doses based on patient cerebral spinal fluid (CSF). However, HIV patients take these drugs for the rest of their lives and levels could accumulate in cells over a period of years to exceed that of CSF. Levels of ABC in brain homogenates from ABC-treated mice support this (There are some caveats pertaining to our results. The blood brain barrier hinders penetration of HAART drugs into the CNS, meaning that the doses used to examine the effect of HAART drugs ort this . Therefoin vitro culturing of fetal astrocytes may not accurately reflect astrocytic function in vivo, we want to validate our results by using an in vivo model exposing mice to these HAART drugs.The use of HAART drug combinations instead of individual compounds means that the contributions of a specific component cannot be discerned. Since HIV patients take antiretroviral compounds as combinations, we decided to focus the scope of this manuscript accordingly. Interesting future directions include determining if individual antiretroviral drugs or classes are the sole contributors to our astrocyte senescence and dysfunction as well as determining if these adverse effects can be pharmacologically attenuated. In addition, since in vivo, our studies support HAART-induced cellular senescence as a mechanism implied in HAND development. In concordance with our results, clinical trial studies have indicated that drugs with greater CNS penetration resulted in impaired neurocognitive performance, even though HIV was suppressed better (While it is likely that other factors including HIV gene products and drugs of abuse may also contribute to the pathogenesis of HAND d better , and thad better . These rOur data demonstrate that HAs senesce in response to a combination of the HAART drugs ABC–3TC–RTV. This has implications for senescence in the CNS contributing to the neurological problems in patients with HAND. Changes in mitochondria, metabolism, and the secretory profile observed in this study suggest that these are potential targets for therapeutics, which could mitigate HAND.JC conceived and performed the experiments and wrote the manuscript; LD, JW, and FT contributed to the experiments; and CT conceived the experiments and helped write the manuscript.The 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.


ARHGEF39) represent new putativerisk factors for SLI. Furthermore, we identified 3′UTR regulatory variantsacross autism, schizophrenia and bipolar disorder NGS cohorts demonstrating theirimpact on neurodevelopmental and neuropsychiatric disorders. Our findings show theimportance of investigating non-coding regulatory variants when determining riskfactors contributing to neurodevelopmental and neuropsychiatric disorders. In thefuture, integration of such regulatory variation with protein coding changes will beessential for uncovering the genetic causes of complex neurological disorders and thefundamental mechanisms underlying health and disease.Understanding the genetic factors underlying neurodevelopmental and neuropsychiatricdisorders is a major challenge given their prevalence and potential severity forquality of life. While large-scale genomic screens have made major advances in thisarea, for many disorders the genetic underpinnings are complex and poorly understood.To date the field has focused predominantly on protein coding variation, but giventhe importance of tightly controlled gene expression for normal brain development anddisorder, variation that affects non-coding regulatory regions of the genome islikely to play an important role in these phenotypes. Herein we show the importanceof 3 prime untranslated region (3'UTR) non-coding regulatory variants acrossneurodevelopmental and neuropsychiatric disorders. We devised a pipeline foridentifying and functionally validating putatively pathogenic variants from nextgeneration sequencing (NGS) data. We applied this pipeline to a cohort of childrenwith severe specific language impairment (SLI) and identified a functional,SLI-associated variant affecting gene regulation in cells and post-mortem humanbrain. This variant and the affected gene ( Great strides have been made inunderstanding the genetic factors contributing to these disorders by screening largecohorts of affected individuals using next generation sequencing (NGS) approachessuch as whole-exome sequencing (WES) and more recently, whole-genome sequencing(WGS). However, for many neuropsychiatric disorders, the underlying genetic causescan be convoluted sometimes involving rare variation across a large number ofcausative genes, small contributions from common variants or no clear genetic causesdespite high heritability of the disorder. As such, neuropsychiatric andneurodevelopmental disorders currently suffer from a high degree of missingheritability that demands new and different approaches when attempting to identifyand assess genetic variation that could contribute to these disorders.Neuropsychiatric and neurodevelopmental disorders affect more than 15% of thepopulation5 due to its ability to rapidly and cheaplysurvey the protein coding portions of the genome. However, by design WES only surveysaround 1% of the total genome sequence meaning that the majority of genomicvariation in each individual cannot be assessed via this method. The importance ofsurveying non-protein coding DNA—which makes up ~99% of the humangenome—is becoming apparent with the growing popularity of WGS studies. WGS hasnow successfully been used to identify hot-spots in the genome that are particularlysusceptible to de novo mutations,6mutation parent-of-origin effects,7 novelgenes underlying intellectual disability8and the prevalence of disruptions of non-coding regulatory regions in autisticindividuals.11 These studies make itclear that we must take the contribution of non-coding variation seriously if we areto fully understand the genetics of neuropsychiatric and neurodevelopmentaldisorders.WES has been a recent method of choice for uncovering causes of neuropsychiatricdisorders such as intellectual disability and ASD15 Regulatory variants that result in significantlyreduced gene expression could mimic loss of function coding mutations. Conversely,given that for many genes too much expression or expression at the wrong place ortime can be as detrimental as gene loss, mutations that increase gene expression orchange its timing/location can also contribute to disorder.17 Altered gene expression patterns are a commonfeature of neuropsychiatric disorders such as ASD and SCZ and in some cases thesechanges in gene expression can be linked directly to genomic/pathogenicvariation.20 This evidence points to a strong link between aberrant geneexpression and neurological disorders, suggesting that variants in non-codingregulatory elements are excellent candidates to fill in some of the missingheritability of neuropsychiatric and neurodevelopmental disorders.Within non-coding regions of the human genome, regulatory elements such as promoters,enhancers and untranslated regions are strong candidate regions for pathogenicvariation because they direct how much, when and where a gene is expressed. Variationin regulatory regions such as single nucleotide variants (SNV’s) or smallindels, as well as larger scale copy number variants have all been shown tosignificantly affect gene expression.24 and have beenimplicated in a range of neurological disorders. Sequencing of genes involved intinnitus, Parkinson’s disease, Tourette’s syndrome and ASD have linkedcommon single-nucleotide polymorphisms (SNPs) and rare variants in 3′UTRregions to these disorders.25 In addition, a number of key considerations makethese regions strong candidates for pathogenicity and highly amenable to study.First, 3′UTR regions are found at the end of all protein coding genes in thegenome and are comprehensively annotated26making their identification routine. Second, 3′UTR regions directly affect theamount of protein produced from a gene transcript via interactions with smallregulatory RNA molecules known as microRNAs. Because these microRNA interactions arebased on complementary nucleotide pairing with short sequences (7–20 bp)in the 3′UTR regions, they can be accurately predicted from sequence data in ahigh throughput manner. Finally, the functional effects of observed variants can betested in the lab using simple, scalable assays. Functional testing to show thatputative causative variants have cellular consequences is crucial for determiningpathogenicity of both coding and non-coding variation and the potential scalabilityof these assays makes testing microRNA binding site (MBS) variants particularlyattractive for large-scale NGS studies.To this end, we chose to investigate non-coding variation in an NGS cohort, focusingspecifically on a well-characterised regulatory region that strongly influences geneexpression: the 3 prime untranslated region (3′UTR). 3′UTRs of genes havelong been considered likely candidates for pathogenic mutations28 Despite strong evidence for genetic underpinnings oflanguage impairment, efforts to identify causative factors via linkage andassociation studies have found multiple genomic regions, all predicted to have smalleffects.31 Thus, like many otherneurodevelopmental disorders, SLI seems to have a heterogeneous set of genetic riskfactors contributing to the phenotype. We used WES data from a cohort of 43 affectedchildren with severe SLI32 to demonstratethat it is possible to identify putative causative variants in non-coding 3′UTRregions using NGS data sets. Importantly we showed that variants can have directfunctional consequences for gene expression by affecting miRNA binding sites, makingthem strong candidates for pathogenicity. To show the wider relevance of this classof variation we demonstrated that non-coding 3′UTR variants that disrupt miRNAbinding sites are found across a range of neurological phenotypes, emphasising theirpotential relevance to neuropsychiatric and neurodevelopmental disorders.Given all these factors, we designed an approach for using NGS data to identify andfunctionally test the effects of variation in the 3′UTRome. Herein we show theefficacy of this method using WES data from a cohort of children classified as havinga neurodevelopmental disorder: specific language impairment (SLI). SLI, the failureto acquire age appropriate language skills in the absence of any explanatory factors, affects up to 8% of school agechildren.etal.32 Briefly, for sequencing,the exome was captured by SureSelect Human All Exon version-2 50 Mb kit, sequenced using the SOLiD series 5500xl DNAsequencing platform  and called via thestandard BWA-GATK pipeline, followed by quality filtering as described by Chenet al.32 Ethical agreementand informed consent from participants were obtained as describedpreviously.29Full methods on WES sequencing and variant calling are described by Chen 33 To identify WESvariants that were overlapping with these microRNA sites we used the BEDToolsintersectBED function.34Genome-wide coordinates (hg19) for MBSs were downloaded from the Targetscan 6.2database.et al.32 SLI exome study are deposited at The Language Archive(TLA: https://corpus1.mpi.nl/ds/asv/?0), a public data archive hosted bythe Max Planck Institute for Psycholinguistics. Data are stored at the TLA underthe node ID: MPI2010433#, and accessible with a persistent identifier:https://hdl.handle.net/1839/00-0000-0000-001E-AD41-2@view.Access can be granted upon request. TLA content is also visible from the DataArchiving and Networked Services (DANS) database, the Dutch national organizationfor sustained access to digital research data.The primary data for the Chen AgeI and EcoRIrestriction sites in the pLKO.1 expression vector and the sequences were confirmed by Sanger sequencing.To generate the microRNA expression constructs for miR-215, -342 and -346, regionsencoding the primary transcripts were PCR amplified using the primers listed in35 In such hybridconstructs, the sequence of the shRNA replaces the sequence that creates thestem–loop of the endogenous miR. Because we knew that miR-342 wassuccessfully expressed  of the miR-342stem–loop. The miR-433 stem–loop was amplified using primerscontaining XbaI and SalI restriction sites . As such an alternativeapproach was undertaken. MicroRNA primary transcripts have been successfully usedto drive expression of synthetic short hairpin RNA (shRNA) to facilitate geneknockdown.xpressed ,we inse37 Briefly, oligonucleotides containing twohigh-sensitivity binding sites for the cognate miRNAs were designed. To clone thereporter cassettes, sense and antisense oligonucleotides were generated andannealed to each other to form overhangs compatible with KflI restrictionsites—allowing directional cloning into compatible sites. Oligonucleotidesused for the generation of the reporters are listed in 'Positive control' reporter constructs were used to confirm that allcloned microRNAs were expressed and able to regulate gene expression byinteracting with ideal target sites in a reporter assay . PositivXbaI and FseI restriction sites.Vectors carrying the alternative allele or with a deletion of the MBS weregenerated using the QuickChange Site-Directed Mutagenesis kit (AgilentTechnologies) following the manufacturer's instructions and using the primersin The 3′UTR regions for each candidate gene spanning the patient identifiedvariants  were cloned intothe pGL4.24 luciferase expression vector  as reporterconstructs. Control human genomic DNA was used to PCR amplify the regions of interest (using the primers in 40 All the experiments were carried out using cells grownin Dulbecco's modified Eagle's medium (Invitrogen) media supplementedwith 10% fetal calf serum  and2 mm penicillin/streptomycin. Cells were kept for theentire length of the experiments at 37 °C in the presence of 5%CO2. Transfections were performed using GeneJuice (Novagen)following the manufacturer's instructions.We performed the reporter assays in human HEK293 cells and for comparison in amouse neuronal cell line: Neuro2A (N2A) cells. Cell lines were obtained from ATCC and are routinely screened for mycoplasmacontamination. Both cell lines are a suitable cellular model as they are easy toculture and reach very high transfection efficiency. The protein machinery thatmicroRNAs use to regulate gene expression is ubiquitous and we and others havepreviously shown that this machinery is functional across different celltypes.4 cells were seeded in each well of a 24-well plate(60–70% confluence) 24 h before transfection. Reporterconstructs were co-transfected into cells alongside the microRNA expression vectorand a Renilla reporter (pRL-TK) for internal normalisation. Forty-eight hourspost-transfection, firefly luciferase and Renilla luciferase activities weremeasured as per the manufacturer's instructions .3.0 × 105 HEK293 cells were seeded in each well of a six-wellplate (60–70% confluence) 24 h prior to transfection of themicroRNA (miR-215) expression vector (4 μg). Forty-eight hourspost-transfection, cells were lysed in lysis buffer  at 4 °C for10 min and centrifuged at 10 000 g for 30 minat 4 °C, allowing cell debris to be pelleted and discarded. Westernblotting was performed as described previously.41 Proteins were detected using primary antibodies for60 min at room temperature or at 4 °C overnight. Secondaryantibodies were applied for 30–60 min at room temperature. Antibodieswere used as follows: ARHGEF39  at 1/2000 concentration,B-actin  at1/2000 concentration, secondary anti-mouse HRP-conjugated antibody  at 1/5000concentration and secondary anti-rabbit HRP-conjugated antibody  at 1/5000concentration. Densitometry was performed using the Imagelab 5.2.1 (Bio-Rad)program to calculate arbitrary units reflecting relative protein expression levelsfor ARHGEF39 and β-actin.3.0 × 10http://www.gtexportal.org; GTEx Analysis Release V6p) to determineassociations between individual SNPs and the expression of their correspondingprotein coding genes. We assessed genes for single-tissue eQTL values andgenerated the multi-tissue eQTL comparison plot using the online ‘GeneAssociation’ tool. Plots for individual brain regions were generated usingthe ‘Test your own’ tool. Descriptions of the association andstatistical analyses generated by GTEx can be found in the relevantreferences.43P-values are calculated for single-tissue associations and given thatassociations were generated for 44 tissues, using a stringent multiple testingcorrection (Bonferroni), P-values must be P<1.13e-3 to beconsidered significant.We utilised the Genotype-Tissue Expression project portal (GTEx)  from these data. For themajority of 3′UTRs in the genome (62.7% of 3′UTRs annotated byRef-Seq), the first ~200 bp had a read depth ⩾10 allowing the reliableidentification of variants in this region , indicating that theyhave a population frequency of less than 0.00082%. The presence of theserare variants in each of the relevant probands was confirmed with bi-directionalSanger sequencing  were not genotyped in the SLIC (SLI Consortium) data set and thuscould not be assessed. However, three of the five common SNPs were directlygenotyped or imputed from the SLIC data set (rs72727021 (imputed), rs383362(genotyped) and rs1049232 (genotyped)) and thus we could use these data for acandidate association analysis. Genotype data were available for 983 parents andchildren from these 285 SLI families. As previously described, variousquantitative measures of language-related abilities were available for children inthis cohort.48 Measures ofexpressive (ELS) and receptive (RLS) language were obtained using the ClinicalEvaluations of Language Fundamentals (CELF-R).49 Verbal IQ (VIQ) and performance IQ (PIQ) were assessedusing the Wechsler Intelligence Scale for Children50 and reading and spelling ability were measured with theWechsler Objective Reading Dimensions (WORD).51 In addition, the phonological short-term memory ofadults and children was assessed with a 28-item test of non-wordrepetition.52 Association wasassessed within and between family units using the QFAM test in PLINK. Thisquantitative test of association employs an adaptive permutation procedure toaccount for the dependence between related individuals. One SNP (rs72727021),located in the 3′UTR of the ARHGEF39 gene , was associated with the non-word repetition measure in theSLI cohort (To determine the relevance of the common (SNP) variants to SLI we took advantageof genome-wide SNP data from the SLI Consortium (SLIC) sample. This data setcomprised 285 SLI families recruited from five centres across the UK; The NewcomenCentre at Guy’s Hospital, London ; the Cambridge Language and Speech Project (CLASP); the Child Life andHealth Department at the University of Edinburgh; the Department of Child Healthat the University of Aberdeen; and the Manchester Language Study. × 10−3). The alt53 and the Colorado LearningDisabilities Research Centre (CLDRC) data set.54 Although no significant association was found in thesedata sets, a lack of support from these cohorts may reflect variations in sampleascertainment since the SLIC population was specifically selected for severelanguage phenotypes, or the differences in tests used to generate the languageability metrics. Further investigations in specifically selected samples will beneeded to clarify the genetic association between rs72727021 and languagephenotypes.We sought to find supportive evidence for the association between rs72727021 andnon-word repetition in other data sets; however, to our knowledge a replicationdata set does not exist. We nonetheless queried the association in two independentdata sets: the Avon Longitudinal Study of Parents and Children (ALSPAC)cohortin silico predictions,we first set out to functionally validate the predicted interaction between these3′UTR-binding sites and their cognate microRNAs using a reporter assay. Wecloned an approx. 300–400 bp region from each genes 3′UTR,spanning the predicted miRNA-binding site  in these reporter assays,suggesting they are not functional binding sites (From our exome data we have identified four candidate variants located in MBSsthat may be related to the SLI phenotype: one common variant (rs72727021) that isassociated with non-word repetition in the SLI cohort and three rare (private)variants. Because these MBSs were identified via ing site .This wang sites . As suchARHGEF39 is predicted to berecognised and bound by both miR-215 and miR-192. The seed sequence for thesemicroRNAs are identical and both microRNAs have the same confidence score (aspredicted by Targetscan), suggesting that they are equally able to bind this site.Given this equivalence, we used miR-215 to demonstrate the functionality of thissite. The ARHGEF39 3′UTR reporter containing the reference alleleof the rs72727021 SNP was significantly downregulated by miR-215   . This mil assays . Tosuppscohort . No signTaken together, these findings show that we have identified a common non-codingSNP with a significant association to SLI and a functional effect on geneexpression in both cell models and the human brain. We believe these datarepresent the first time an SLI-associated SNP has been reported to have directfunctional consequences.de novovariation in large cohorts and for which variant call files were made availableonline. These studies included whole-exome and WGS data sets from cohorts ofbipolar disorder, SCZ, ASD and a study investigating parent-of-origin signaturesof de novo mutations.57 We determined the number of variants in3′UTR regions that altered MBSs via intersection of these variants withTargetscan coordinates (as described above) and identified 37 MBS variants intotal. In all studies tested at least one MBS variant was found, and up to 12de novo variants could be identified per cohort  in languagedisorder. Little is currently known regarding the ARHGEF39 gene, but it is amember of the ARHGEF family of Rho guanine nucleotide exchange factors. Theseproteins act as molecular switches to regulate diverse processes includingtranscriptional regulation, cell migration, cell growth and dendriticdevelopment.60 Other members of thisfamily have previously been implicated in language impairment(ARHGEF19),29intellectualdisability (ARHGEF6)61 andmoderate intellectual disability with speech delay (ARHGEF9).4 Future studies into the expression pattern and function ofthis gene may reveal a potential role for ARHGEF39 in the development oflanguage relevant circuitry in the brain.This work represents the first report of functional effects for a non-coding SNPassociated with SLI and highlights a possible role for the rs72727021 variantand/or the de novo variants inMBSs that may contribute to the aetiology of these neuropsychiatric disorders current WES platforms do not sequencethe entire 3′UTR, we have shown that we can extract functional variants fromexome sequence data using our SLI cohort. Furthermore, we have shown that otherexisting WES data sets investigating SCZ and ASD contain isorders. To date56 selection criteria were conservative , orwhen variants were only included from a small number of candidate genes,56 at least one 3′UTR-MBS variant wasidentified per cohort, suggesting that this class of variants will be highlyprevalent across WGS studies. Further supporting the relevance of these variants tophenotypes, the MBS variants we identified in these cohorts were often located in the3′UTRs of genes previously implicated in their respective disorders. Forexample, in the bipolar cohort four MBS variants across six probands were identifiedin a leading candidate gene, ANK3.56 In ASD cohorts, MBS variants were identified inBRD12 andCHD2,9 both of which havebeen previously linked to autism via identification of putatively pathogenic denovo protein-coding mutations and copy number variants.64 Indeed, taken together, these findings argue forexamination of the 3′UTR regions of candidate genes across a range of disorderswithin existing NGS data sets. Our findings suggest it is likely that new3′UTR-MBS variants will be identified via this approach that may help toexplain some of the missing heritability of neurodevelopmental/neuropsychiatricdisorders.Studies of complex disorders are increasingly making use of WGS, and including ourpipeline when assessing WGS data from large cohorts will allow unprecedentedidentification of functional, non-coding risk factors. In the WGS data sets wesurveyed  even whe66 reinforcing the importance of functional testingwhen attempting to link genetic variation to phenotypes. A major strength of ourapproach is that it provides a simple, high throughput method for validating thefunctional, molecular consequences of non-coding 3′UTRome variants. Because thefunctionality of a microRNA-3′UTR interaction can be determined via a simpleset of reporter gene assays in any cell model, this approach facilitates highthroughput and reliable assessment of the consequences of identified 3′UTRvariants.NGS has brought astonishingly rapid advances in the high throughput identification ofvariants related to phenotypes. Unfortunately our ability to determine which variantshave functional consequences has not kept pace. Yet, if a genetic variant does nothave molecular consequences for a gene or a cell, it is unlikely that it will becausative or pathogenic, making functional analysis crucial. Algorithms are widelyused to predict functional/pathogenic consequences; however, these have very highfalse-positive/negative ratesIn summary, we report an accessible, rapid and biologically relevant method forassessing non-coding variation that adds significantly to our ability to identifycausative variants from NGS data—a major challenge facing the future ofgenomics. Neuropsychiatric disorders suffer from a high degree of missingheritability and one of the ways we can address this issue is to look beyond a‘protein-centric’ view of the genome and also address the relevance ofnon-coding variation. Adding this simple pipeline to the standard WES/WGS toolkitis expected to reveal a wealth of functional variation in largely overlookednon-coding regions of the genome and will help to identify new links between genesand disorders. Furthermore, by not stopping at identification but performing rigorousfunctional characterisation of the molecular consequences of these variants, we willshed new light on not only the causes of neurological disorders but the fundamentalmolecular mechanisms underlying health and disease.


Using 16 S rRNA gene clone library analysis we revealed that the bacterial community is dominated by Cyanobacteria, Alphaproteobacteria and an unusually high proportion of Chloroflexi, followed by Armatimonadetes and Planctomycetes, and is therefore unique compared to other living examples. Macroinvertebrates are sparse and snails in particular are disadvantaged by the development of debilitating accumulations of carbonate on their shells, corroborating evidence that stromatolites flourish under conditions where predation by metazoans is suppressed. Our findings constitute a novel habitat for stromatolites because cool-temperate freshwater wetlands are not a conventional stromatolite niche, suggesting that stromatolites may be more common than previously thought.Stromatolites are the oldest evidence for life on Earth, but modern living examples are rare and predominantly occur in shallow marine or (hyper-) saline lacustrine environments, subject to exotic physico-chemical conditions. Here we report the discovery of living freshwater stromatolites in cool-temperate karstic wetlands in the Giblin River catchment of the UNESCO-listed Tasmanian Wilderness World Heritage Area, Australia. These stromatolites colonize the slopes of karstic spring mounds which create mildly alkaline (pH of 7.0-7.9) enclaves within an otherwise uniformly acidic organosol terrain. The freshwater emerging from the springs is Ca-HCO These microbial accretions are the oldest evidence for life on Earth6 and debates continue on whether they evolved first on land or in the ocean10. Fossilized stromatolites and similar accretionary microbial mats have provided intriguing microbial archives for over a century12, revealing that microbialites were abundant in the shallow late-Archean and Proterozoic oceans, but declined with the emergence of multicellular life in the Cambrian13. The evolution of grazing metazoans has therefore been suggested as a primary cause of stromatolite decline during the history of life on Earth. However, the role of metazoans in limiting stromatolite formation has been questioned15, and a living example for the co-occurrence of stromatolites and benthic macroinvertebrates has recently been reported16. Evidence from gene sequencing also suggests that microbialites support diverse and distinct active eukaryotic communities which may influence microbialite structure17.Stromatolites are a form of microbialite with repetitive, laminated structures of biologically  mediated mineral precipitation19. Well-known living examples are shallow marine stromatolites in Hamelin Pool, Shark Bay, Western Australia6, and the shallow subtidal stromatolites in Highborne Cay, Bahamas10. Other occurrences include (hyper-) saline lacustrine environments such as Storr’s Lake, Bahamas12, a hyper-saline lake of the Kiritimati Atoll, Central Pacific20, high-altitude Lake Socompa, Argentina15, and supratidal pools along the coastline of South Africa21. However, stromatolites growing in low-salinity, low-temperature freshwaters have also been recognized at localities such as: Ruidera Pools Natural Park, Spain22, Pavilion Lake, British Columbia, Canada23, karst-water creeks in Germany and France25, cenote lakes in south-eastern mainland Australia26, and tufa depositing streams in SW Japan27.Modern living stromatolites are rare but occur in diverse habitats which are often subject to extreme conditions inhospitable to other life forms28, because the water is neutral to mildly alkaline (pH ~7–8), creating exceptional pH gradients across the boundary between the wetlands and surrounding acidic blanket bogs (pH ~4–5). This is striking because the majority of surface waters in south-west Tasmania are strongly acidic due to the high humic content of the soil29. We refer to these features as ‘peat-bound karstic wetlands’, because they comprise ‘islands’ of peat-free ground within otherwise monotonous organosol terrain of karstic limestone and dolomite valleys . Previously interpreted as ephemeral disturbance features due to burning of the peat with resultant exposure of underlying alkaline substrates, water chemistry data lead us to re-interpret the peat-bound karstic wetlands as spring-fed groundwater-dependent ecosystems.The Giblin River catchment and certain others in south-west Tasmania, Australia, contain significant concentrations of unusual wetlands comprising poorly drained, sparsely vegetated sandy to gravelly flats of variable size and shape Fig. . Older l30. We have studied the microbial structure of the Giblin River stromatolites and characterize the physico-chemical conditions of their habitat to determine environmental parameters that control and allow modern freshwater stromatolite formation in this region.Recent visits to peat-bound karstic wetlands in the Giblin River catchment revealed that their surfaces are colonized by aggregations of stromatolites Fig. . There a−1 .Stromatolites colonize the lower, peripheral slopes of the Giblin River spring mounds, forming smooth mats of yellowish and greenish, globular structures growing on the wetted surface of tufa barriers Fig. . The str SI Fig. , reveali−1. The temperature indicates negligible geothermal heating (10.6–14.4 °C). These waters are Ca-HCO3 dominated, followed by Cl, Mg, Na and SO4   is mildly alkaline freshwater with a pH of 7.5–7.6, zero salinity, and electrical conductivity of 618–640 µS cm3 under conditions of elevated pCO2. As the water flows downslope towards the edge of the mound, it equilibrates with atmospheric pCO2 (degasses) resulting in increasing saturation indices (SI), causing precipitation of CaCO3. As Ca precipitates and the water mixes with acidic peat water (pH 5.3) towards the margin of the mound, saturation indices decrease markedly and waters are under-saturated with respect to calcite, aragonite, and dolomite. We also noted that dolomite saturation features a similar behavior compared to calcite and aragonite SI across the mound, but indices are twice as high towards the slopes of the mound (SI > 2) . Aragonite or biologically formed amorphous calcium carbonate would result in different Raman spectra compared to crystalline calcite32. Our findings are consistent with the observed behavior of typical tufa-depositing springs elsewhere in the world33, although biotic processes  may contribute at some level to the observed chemical evolution of the water flowing across the mound.The transect across the largest mound revealed a distinct pattern of physical and chemical water properties from the centre of the mound towards the peatland Fig. . The spr37. Chloroflexi phylotypes formed two major clades , including one that branched within class Chloroflexia and another that represented a branch within the class Anaerolineae. The latter clade was distant from the cultured representatives of the Anaerolineaceae38, but were most similar to taxa from biofilms of rock surfaces and mineral springs such as those described by Tomcyzk-Zak et al.39 and Headd & Engel40. The community composition data also implied the presence of a wide diversity of heterotrophic species normally found in abundance in oligotrophic freshwater ecosystems including microbial mats and stromatolites42. These taxa include members of the genera Caulobacter, Hyphomicrobium, families Hyphomonadaceae, Sphingomondaceae, Cytophagaceae, Saprospiraceae, and the phyla Planctomycetes and Armatimonadetes. Potentially these bacteria may depend on carbon derived from cyanobacterial primary production or could assist in decomposition of the cyanobacterial mats. Overall, based on the OTUs detected, there was limited evidence of strictly anaerobic bacteria being also present in the samples, which is consistent with the samples analysed being derived from a largely active photosynthetic mat growing on tufa. Overall, the OTUs detected were generally distinct from cultured and uncultured bacterial taxa and showing no close correspondence to OTUs detected in other stromatolite systems, including freshwater stromatolites22 and lake microbialites43  of the Giblin River stromatolite bacterial community belonged mainly to Cyanobacteria, Chloroflexi, Armatimonadetes, Alphaproteobacteria and Planctomycetes Fig. . CyanobaGiven the uniqueness of the microbial community composition within Giblin River spring mound stromatolite formations, they could derive from local species present in adjacent spring water, soil and rock biofilms. However, to date, none of these ecosystems have received any detailed microbiological study and thus a complete comprehension of the microbiology of the stromatolites will require analysis of the surrounding Giblin River spring and wetland system.15. We have therefore investigated macroinvertebrate abundance at the Giblin River spring mounds. Macroinvertebrate sampling indicates that the Giblin River spring mounds host a sparse assemblage of invertebrate taxa most of which are not yet identifiable to known, described species , the Planorbidae Glyptophysa gibbosa, and 5 Hydrobiidae: Phrantela singularis, Phrantela angulifera, Austropyrgus pisinnus and 2 unidentified Phrantela spp., with A. pisinnus and P. singularis being most abundant. The relative abundance of other snail species is low. Many snails, both live and deceased, were affected by carbonate encrustations on the shell – in some cases the original form of dead shells were entirely obscured by bulbous masses of accreted minerals , and lies within the UNESCO-listed Tasmanian Wilderness World Heritage Area. This region experiences cool temperate maritime conditions dominated by ‘roaring forties’ weather off the Southern Ocean . The Giblin River occupies a broad syncline where dissolution of Ordovician limestone has produced an undulating, low-relief surface a few tens of metres above sea level. This surface is largely covered by Tertiary siliceous alluvial gravels and Holocene blanket bogs, a form of ombotrophic peatland which mantles large tracts of western Tasmania. The vegetation is dominated by grassy to shrubby moorland, generally considered a successional disclimax related to earlier indigenous burning. The catchment is isolated from settled areas to the east (>100 km) by rugged topography and its natural values are poorly documented.Within the Giblin River catchment occur peat-bound karstic wetlands that are regionally distinctive due to the presence of prominent spring mounds Fig. . The mou−1 between 220 and 1200 cm−1. The spectrum was baseline corrected to remove the background fluorescence.A cross-section of a representative stromatolite sample collected near site GR2 during the first visit , and salinity using a salinity refractometer. Samples for ion chemistry were collected in 50 ml centrifuge tubes after 0.45 µm filtration. Samples for bicarbonate titrations were not filtered. All water samples were stored below 4 °C until further analyses. Major anions and cations were analysed by ion chromatography (Dionex ICS-3000) at the Australian Centre for Research on Separation Science (ACROSS), University of Tasmania. Bicarbonate and total alkalinity were determined by titration with HCl. Water samples were also analysed for water isotopes (δ18O and δ2H) using a Picarro Laser Cavity Ringdown Spectrometer at the Australian Antarctic Division, Hobart.Water sampling was undertaken at four locations within the wetland GR1 to GR4) in December 2015 and at two locations in August 2016  were calculated using the software Geochemist’s Workbench® and are defined as SI = logIAP/K, where IAP is the ion activation product and K the equilibrium constant.http://hannonlab.cshl.edu/fastx_toolkit/index.html). Reads were then quality filtered and denoised in Mothur v. 1.35.151. Sequences with a mean PHRED score of >30 and containing bases of scores <8 were discarded. Alignment, clustering and chimera removal was then performed using UPARSE52. After manual inspection there was still potential evidence of chimeras amongst OTUs despite use of UCLUST as a result clusters were further checked for chimeras using DECIPHER in short sequence mode53. This approach was useful in flagging potential chimeras for assessment. Singleton reads were excluded from further analysis. The remaining OTUs were classified using PyNAST 1.154 against the Greengenes database (March 2013 version)55. From the two samples 152145 and 127009 reads were obtained after filtration and grouped collectively into 1063 OTUs at the 97% level. Both samples were highly similar sharing 61.4% of the OTUs. Of the OTUs 208 were designated unclassified. Manual alignments were performed to assess their phylogenetic placement by utilizing a combination of Greengenes database and a custom NCBI 16 S rRNA gene sequence database. Of these OTUs 21 were found to be chimeric and thus culled. This polished dataset was then used to compare the taxonomic makeup with other stromatolite data available in the NCBI database57.Two microbial samples of stromatolitic smooth mats were collected in 50 ml centrifuge tubes near site GR2 during the first visit (December 2015), and stored below 4 °C until further analysis. DNA extraction was performed on both samples using the DNeasy PowerSoil kit  and 16 S rRNA gene sequence analyses were subsequently performed using services and facilities at the Australian Genome Research Facility (AGRF). PCR primers used were for the V1 to V3 domain regions of the 16 S rRNA gene including 27 F (AGA GTT TGA TCM TGG CTC AG) and 519 R (GWA TTA CCG CGG CKG CTG). Following purification of the amplicons dual indices and adapter primers were added to the amplicons using the Nextera XT Library Prep kit . Following denaturation and pooling the libraries were sequenced using the Illumina MiSeq platform according to standard protocols generating 300 bp pairended reads. Sequence processing and assessment was performed by joining FastQ files using FastX  were applied, depending on the substrate conditions. Core sampling consisted of applying a 50 ml syringe (top removed to form a cylinder) to the substrate within a 20 cm46.Samples were all preserved and sorted to family (or genus) under a dissection microscope. Mollusca were further identified to species using microscopy and dissection, current keys and descriptionsRepresentative OTU sequence data were deposited in the NCBI GenBank database under accession numbers KX903306-KX904341.Supplementary Information