Wedemonstrate that light can indeed drive lithiation changes in LixV2O5 while maintainingcharge neutrality, possibly via a combination of faradaic and nonfaradaiceffects taking place in individual particles. Our results providean addition to the photobattery mechanistic model highlighting thatboth intercalation-based charging and lithium concentration polarizationeffects contribute to the increased photocharging capacity.Photobatteries, batteries with a light-sensitive electrode,haverecently been proposed as a way of simultaneously capturing and storingsolar energy in a single device. Despite reports of photochargingwith multiple different electrode materials, the overall mechanismof operation remains poorly understood. Here, we use  However, the inconsistency of daily insolationmeans that to ensure autonomous operation, the solar energy harvestingaspect must be combined with an energy storage system to providea continuous source of electricity during the operation cycle of thedevices.8 Most solar cells have open circuit voltages of 0.6–1.0V,9 which makes them unsuitable to be useddirectly with Li-ion batteries (LIBs) without dc-to-dc power converters,or stacking.8 Similarly, at the utility scale,the output of a string of solar modules requires expensive power electronicsto couple the energy-harvesting and storage infrastructures. Muchwork has been done on tackling this problem such that ohmic lossesand packing inefficiencies between the two devices can be reduced;however, ongoing manufacturing challenges, which ensure performancewithout increasing the balance of plant costs, remain.8 An ideal solution would be the development of photoelectrodesthat can harvest solar energy and store it natively without the needfor energy converters.12 Such a technology could conceivably be implementedat the device scale or in off-grid rural settings, where autonomousoperation and low maintenance are particularly desirable.Low-power consumption devicessuch as wireless sensor networks are playing an increasingly importantrole in the 21st century.14 as well as more integrated two-electode systems that have been appliedto Li-ion,17 Zn-ion,18 and Ag-ion batteries.19 Two-electrode solar batteries rely on materialsthat can simultaneously harvest and store energy. In this configuration,photocharging has been demonstrated with organohalide perovskites,16 MoS2,20 and organic materials,22 with many moreproposed or in early stages of study. One of the most promising materialsthat has emerged for photobatteries is V2O5,which has demonstrated the ability both to harvest solar energy andto store it in the form of a Li-ion battery (LIB),17 a Zn-ion battery,21 and even a photoactiveZn-ion capacitor.20 However, even withLixV2O5, the overallpower conversion efficiencies of photobatteries have remained low,particularly in terms of the maximum charge state that can be achievedunder illumination. To begin to overcome this, a better understandingof the photocharging mechanism is required.Severaldifferent strategies have been proposed to combine energyharvesting and storage. These include three-electrode photobatterycells, in which the energy harvesting and storage units are compartmentalized,19 Photoexcitedcharges are generated under illumination and then separated by thepresence of hole/electron transport layers neighboring the photoactiveelectrode material. The electrons are transported to the anode torecombine with Li+ ions to form Li(0), triggeringthe release of intercalated ions from the cathode;18 this process, however, is energetically challenging tojustify. An alternative explanation is that the photo-generated electronsreact to form a solid–electrolyte interphase.15 While an attractive idea, it is for instance unclear howthis can explain decrease in impedance observed during cycling underlight.17 Consequently, the question ofwhether photocharging effects are “real” or if the measuredeffects are the result of a series of side reactions and thermal effectsremains unanswered.The hypothesis asto how photobatteries work mechanistically hasbeen founded on that of a heterojunction solar cell.xV2O5 particlesand directly interrogate the photocharging mechanism. We find thatexposure to light for which Ephoton > EBG  results in polarization of theLi concentration within the electrode particles, which is absent withsub-band gap illumination or upon heating of the cell. Our resultsshow that light can drive changes in lithiation of LixV2O5 and indicate hithertounseen surface and subsurface effects in the particles that may allowcharge neutrality to be maintained. More generally, our work demonstratesan optical methodology for robustly interrogating charge dynamicsin photobattery electrode materials, which is essential for theirstudy and optimization.Here, we use optical reflection microscopyto track the lithiationand delithiation in individual 2–15 μm polycrystallineLixV2O5 band edge, and a CMOS detector. The cell is mounted in themicroscope and connected to a potentiostat to control the electrochemistryof the cells while monitoring their optical properties in real time.Like many oxide materials, the visible (400–800 nm) reflectivityof LixV2O5 changesin both spectral position and intensity drastically with the stateof lithiation.24 Specifically, in LixV2O5, the changes in reflectivitywith the lithium fraction are suggested to arise from varying degreesof tilting of the VO6 octahedra and V 3dxy–O 2px/2py orbital overlap in the different lithiation states.26 While such changes in reflectivity with lithiation state are known,the spatial pattern in which they occur on the micrometer scale particles,a first step in understanding the (de)lithiation mechanism, has yetto be established. Wide-field optical microscopy is therefore an idealtool for investigating lithiation dynamics in this system (and manyothers30). The drawbacks of the technique are that it is restricted to probingtwo-dimensional dynamics and particles on the far side of the cellseparator where not all of the material may be active and electrolytedepletion effects may be accentuated (see Supplementary note 1 for mitigation approaches). Furthermore, quantitativelylinking spatial reflection intensities to the material (de)lithiationstate remains challenging using optical microscopy alone, with cautionneeded when comparing interagglomerate behavior.xV2O5 active material (∼90%loading)in a matrix of Super P conductive carbon and a poly(vinylidene fluoride)(PVDF) binder in the custom coin cell geometry shown in xV2O5 particles,the electrodes are modified by mixing the LixV2O5 active material with P3HT and rGO,which act as electron transport layers17 (see Supplementary note 1 for fabrication).We use electrodes made with the LixV2O5 particle, during a discharge (lithiation)/charge(delithiation) cycle. We note that although there is a range of particlesizes on the electrode (which may exhibit a range of charging dynamicsdepending on the size), our studies are limited to 2–15 μmparticles that are relatively isolated from one another (∼5μm separation) for ease of identification and analysis .Upon lithiation, a decrease in reflection intensity (at 650–900nm) is observed, whereas upon delithiation, the intensity of the lightreflected from the particles increases. This in agreement with spectralreflectivity measurements (see Supplementary note 3) that show an increase in the percentage reflectance duringthe charging of bulk LixV2O5 electrodes  at wavelengths of 650–900nm.23 Repeated imaging over multiple cycles, asshown in –2) means we do notexpect large discrepancies in the state of charge between particles.In general, we find that of the 63 particles we image, >85% areelectrochemicallyactive and show similar behavior as detailed in Supplementary note 3).ΔI,is taken as I(ti) and I(to) are normalized intensity images at a given point, ti, and at the start, to, of the cycle, respectively, i.e., when acell is at its maximal state of charge (see Supplementary note 4). The differential imaging technique allows us to removebackground contributions and inhomogeneities in the illumination byisolating and subtracting changes between the images.35 Before calculation of ΔI, the normalizedintensity images are thresholded (see Supplementary note 4) to remove further noise and nonparticle contributions.A negative (positive) differential contrast indicates a decrease (increase)in reflectivity compared to that of the particle in its maximal chargestate.To analyze (de)lithiation under galvanostaticconditions, we determinethe differential intensity contrast across a cycling particle overtime. This quantity, which we denote as ΔI) of theLixV2O5 particleconsidered in 35 Conversely, during delithiation (charge), a front of positiveintensity, corresponding to areas of lithium loss, is observed tomove in the same direction. We note that at the start of the cyclethe differential contrast at the particle center is very slightlypositive (20 min image); however, this is likely due to illuminationvariations or small lateral particle movements (see Supplementary note 4 for correction methods). We collect lightfrom an axial extent of ∼1.5 μm inside the particles;hence, our signals are a projection of the surface and bulk dynamicsof particles (see Supplementary note 5 fora discussion of penetration depth). While particles do have a degreeof positive curvature, given that the entire particle can be placedin focus in a single plane and the particle periphery can sometimesbe brighter than the center, i.e., in opposition to what would beexpected on the basis of signal path lengths alone, we can discountthe significance of such effects. In Supplementary note 4, we repeat measurements in several different focal planesto ensure our observations do not arise from particle curvature orrefractive index mismatch effects, affording qualitatively similarbehavior as in Supplementary note 4). Finally, we note that during lithiation/delithiation LixV2O5 is known to undergo severalphase transitions.37 In our data, we demarcate these transitionsin the potential and time plots of Supplementary note 3), it is not necessarily linear; e.g., there is a smallchange in reflectivity with potential in the δ phase that islikely due to the electronic properties of the underlying phase itself.Furthermore, while the differential intensity over the entire particleand differential voltage with respect to time appear to be correlated(see Supplementary note 6), suggestingour experiment is indeed sensitive to phase boundaries, additionalwork is required to resolve these fully.During both charge and discharge processes, themain changes inlithium concentration are occurring around a central region of theparticle with a distinct shell developing after 50 min of the cycle.After 50 min, the differential contrast dissipates to form a morehomogeneous region across the entire extent of the particle, indicatingthat from this point, changes in lithium concentration occurralmosthomogeneously throughout the whole visible extent of the particle.The behavior displayed in 2O5, we apply the technique to study (de)lithiationunder photocharging conditions. Experiments are repeated in the sameconfiguration as shown in 17 The energy level diagram of the LixV2O5 photocathode under photochargingconditions is shown in 20 To date, some photocharging processes have been carried out underan open circuit condition, which should forbid the motion of the electronfrom the cathode to the anode. However, in these studies,18 a clear increase in cell potential and even additional charge capacityare observed. Here, we endeavor to reconcile these two effects byimaging the Li-ion-containing phase dynamics within individual LixV2O5 particles alsounder illuminated galvanostatic cycling.Having establishedthe ability of optical reflection microscopyto track the intercalation/deintercalation of Li-containing phasesin V–2 intensity at the sample)is used to illuminate the entire electrode. Any reflected light fromthe charging LED is rejected from the camera with appropriate filters,and the imaging is again performed from 650 to 900 nm. The sourceused for imaging is sub-band gap and hence does not induce any photochargingeffects itself.38 To begin, the cells aregalvanostatically discharged to 2.2 V. This is followed by measurementof the open circuit voltage under two conditions: with LED illuminationand without (dark). At open circuit conditions it is assumed thatit is not possible for charges to travel completely from the photocathodeto the Li anode. Therefore, in this configuration, we specificallyimage what we understand to be the initiation of the photochargingprocess, namely the removal of electrons and redox-induced delithiationfrom the LixV2O5 particles.Under photocharging conditions, a 515 nm LED  are plotted for singleLixV2O5 particleswhile measuring the open circuit potential (OCP) without LED illumination.In the absence of LED illumination, little change is observed in thedifferential contrast over time, with just a small degree of brightening(∼5%) of the image concomitant with an increase in the OCPfrom 2.2 to 2.4 V over 500 min. This small change could be a resultof small focus fluctuations and thermodynamic relaxations within thecell. From 500 to 2000 min, both the reflected intensity from theparticle and the OCP remain constant. The particle edges in the differentialimage are slightly negative likely to refractive index mismatchesbetween the particle and matrix.39In Supplementary note 4)) throughout the photocharge.The measured OCP also increases from 2.2 to 2.9 V during 2000 minof light exposure, consistent with previous studies.21 On the basis of our previous observations, an increase in the differentialcontrast suggests that there is a local decrease in the Li-ion concentration,i.e., lithium-poor phase at the particle center, and the dimming atthe periphery indicates an increase in Li-ion concentration at theedges of the particle, i.e., lithium-rich phase. This would implythat light exposure drives lithium from the center to the edges ofthe LixV2O5 electrodeparticles. The behavior can be reproduced over multiple cycles, suggestingthat irreversible side reactions play a limited role in the behavior.Furthermore, changing the photocharging wavelength to 480 nm  resultsin qualitatively similar behavior, tentatively suggesting that theprecise energy of photons does not play a significant role but requiresfurther investigation (see Supplementary note 7).When the battery is exposed to the green LED light,however (photochargingcondition), a significant decrease in the differential contrast (darkening)is observed at the edges of the particles that is concurrent withan increase in contrast (brightening) at the center, as shown in xV2O5, suggesting that the additional elements of a photoelectrodecomposition, namely, the electron transfer layer, play a key rolein the effect. Indeed, repeating experiments without an electron transferlayer (Supplementary note 7) does not resultin behavior like that detailed in 38 A mechanism that has the potential to invoke this effect is thetransfer of photoexcited electrons from LixV2O5 to the P3HT electron transport layer andthe subsequent motion along the induced concentration gradient oflithium ions to the exterior of the particle. Under illumination,we observe the buildup of a Li-rich phase on the exterior of the particles,which we would expect to lead to a lower cell voltage. However, wedetected an increase in OCV ,which leads us to believe that Li ions are leaving the active materialbecause delithiation would correspond to an increase in the stateof charge of the battery. Because under the OCV the electrons cannottravel from the cathode to the anode, a logical explanation wouldbe that the generated electrons and Li ions are stored at the electrodesurface (see the schematic in The results presented above cannot be explained inthe case ofan isolated particle of LiSupplementary note 4 for the definitions of regions). As already determined,the intensity of the core region increases during the photochargingprocess, indicating a reduction in the relative lithium concentration.Conversely, the intensity of the outer ring decreases, indicatinga local increase in the lithium concentration. The gray line is seento increase to a slightly larger magnitude than the gold line decreases,indicating that the sum over both regions (core and outer ring) resultsin a net loss of lithium; i.e., more lithium is lost by the core thanis gained by the periphery. This must imply the loss of lithium tothe volume surrounding the particle, in agreement with our proposedexplanation described above. If lithium is indeed lost to the surroundingelectrolyte, this would necessitate a transfer of charge from theelectrolyte to LixV2O5 for charge compensation, which in turn would originate from thelithium metal anode. The overall effect is that of a genuine rechargemechanism.The net intensity changes of the core region(gray) and edge region(gold) are plotted in the inset of 40 In this work, we observe that during a galvanostaticdischarge when the electrode is illuminated (the requisite conditionsfor photoenhanced operation), the reflected intensity and voltageboth decrease more slowly than when the same electrode is dischargedin the “dark” as shown in “Photo-enhanced” is the term that hasbeen used todescribe the condition in which a cell is controlled via a potentiostat,but with light-induced effects bolstering some performance metricsof the overall device such as the discharge capacity and capacityretention at higher current rates.Supplementary note 8). Nonetheless, our results suggest thatit is the act of closing the external circuit that results in thehomogenization of the previously built-up polarized state across theparticle.To investigate the proposed polarization modelof the photochargingeffect, we repeat the photocharging experiments, and after ∼900min of photocharging under open circuit voltage conditions, we closethe external circuit. As shown in Supplementary note 7). In this case, the charge and discharge curves closely resembleone another regardless of the additional heat. There is a small changein the position of various potential plateaus and gradients, as expectedfrom the increased temperature, but no effects resembling those fromlight are observed. Similarly, concomitant increases and decreasesin the reflected intensity from the particle are observed when chargingand discharging both at room temperature (RT) and at 333 K. This suggeststhat thermal effects can be neglected in the lithium-ion photochargingmechanism in this cell. Additionally, although vanadium oxide materialsare known to undergo a transient phase transition41  under strong pulsedillumination or deep ultraviolet illumination42 , the illumination powers and wavelengthswe use here cannot facilitate this. Indeed, under prolonged lightexposure at 515 nm, we observe no change in the reflectivity of nativeV2O5 films (see Supplementary note 3).To verify that the observations are not drivenby heating effects,we perform galvanostatic charging and discharging from 1.8 to 4.0V at 333 K (60 °C) (39 (see Supplementary note 4 for furtherdiscussion).Finally, to verify that the effect is associatedwith lithium motionand is not an electrochemical or optical artifact, the cells are galvanostaticallycharged to 4.0 V forcing the completion of all delithiation processes,followed by exposure of the cell to the photocharging conditions 5c. No chxV2O5 particles under a variety of electrochemical,photocharging, and thermal conditions. We interrogate the photochargingphenomena reported in previous studies and observe that the processconsists of a buildup of a Li-rich phase on the periphery of the activematerial and a partial delithiation. These effects may result in acombination of faradaic and nonfaradaic charge storage mechanismson the particle exterior. Finally, we demonstrate the dynamics ofsuch electrodes under photoenhanced operation, whereby light may beused to replenish a cell as it simultaneously discharges, allowingfor a greater amount of power to be potentially drawn by an externaldevice. Our results provide a more accurate description of the microscopiceffects of illumination on light-sensitive battery electrode materialsand a methodology for robustly probing microscale charge dynamicsin photobatteries.In summary, we use optical microscopy techniquesto investigatethe (de)lithiation processes in Li22 or layered nanomaterials,20 where both intercalation and conversion reactions may takeplace. While we have focused on one part of the photocharging mechanism,electron transport within the charge transport layers and electrolyteneeds to be investigated further. In terms of using optical microscopyin batteries, obtaining a quantitative readout remains an importantgoal. This may be achieved by converting reflectivity values intorefractive indices , utilizing chemically specific optical probessuch as Raman43 or hyperspectral imaging, correlating optical reflectivitymeasurements with X-ray imaging,45 orcombining optical microscopy with more complex electrochemical toolssuch as the galvanostatic intermittent titration technique to obtainrobust insights into material diffusion coefficients.Future studies should aim to investigatehow the results presentedhere translate to other photobattery materials such as halide perovskites


Heterobasidion parviporum, we have investigated the genetic basis of phenotypic variation among laboratory-designed heterokaryons carrying the same pair of haploid nuclei, but a different mitochondrial genome. Radial growth rate data of thirteen unrelated homokaryotic parents and of their heterokaryotic offspring were combined with SNP data extracted from parental genome sequences to identify nuclear and mitochondrial loci involved in mitonuclear interactions. Two nuclear loci encoding mitochondrial proteins appeared as best candidates to engage in a genetic interaction affecting radial growth rate with a non-conserved mitochondrial open reading frame of unknown function and not reported apart from the Russulales order of Basidiomycete fungi. We believe our approach could be useful to investigate several important traits of fungal biology where mitonuclear interactions play a role, including virulence of fungal pathogens.The mitochondrial and nuclear genomes of Eukaryotes are inherited separately and consequently follow distinct evolutionary paths. Nevertheless, the encoding of many mitochondrial proteins by the nuclear genome shows the high level of integration they have reached, which makes mitonuclear genetic interactions all the more conceivable. For each species, natural selection has fostered the evolution of coadapted alleles in both genomes, but a population-wise divergence of such alleles could lead to important phenotypic variation, and, ultimately, to speciation. In this study in the Basidiomycete  Mitochondria are functionally and genetically dynamic organelles essential to the eukaryotic cell  in host wood , and a non-conserved mitochondrial locus of unknown function and whose distribution is limited to the Russulales order of Agaricomycetes. We believe that our results on mitonuclear genetics of growth rate in H. parviporum could pave the way to similar investigations of mitochondrial DNA-dependent variation of important traits e.g., virulence in fungal pathogens.The present study is an investigation of the genetic basis of mitonuclear interactions in H. parviporum homokaryotic isolates out of thirty initially crossed in our previous study  were estimated by the method of the least squares, using published data of growth measurements of parental homokaryons and their heterokaryotic offspring . Positions of mitochondrial SNP loci were searched in H. irregulare annotated mitochondrial genome (accession NC_024555.1) using BLASTN software (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and published mitochondrial ORF predictions  at the origin of a non-synonymous mutation in isolates Ho9 and Ho32. In brief, the procedure relies on standalone nucleotide blast searches with H. parviporum genomic fragments surrounding each SNP as queries against a custom database including 13,405 open reading frames (ORF) of the related species H. irregulare  published as nc-ORF5 in Himmelstrand et al. . Proteins similar to actual nc-ORF5 translation product were searched using BLASTP software (https://blast.ncbi.nlm.nih.gov/Blast.cgi).Transcripts of the 936 bp-long hypothetical ORF , a large group with high maximal growth rate and low phenotypic variance (107 isolates), and a medium group with high maximal growth rate, but a high phenotypic variance that seems intrinsic to their parental combinations (36 isolates).r2 = 0.55714). In the heterokaryons however, a high QCD is neither explained by a low median growth rate (r2 = 0.26644), or by having one or two parents with a high QCD (r2 = 0.0155). This analysis ruled out the possibility that high level of data dispersion could have experimental cause and rather points at specific heterokaryotic combinations to explain this phenomenon.Dispersion of growth rate data was analyzed both in the heterokaryons and their homokaryotic parents to detect those with high level of data dispersion, and, if so, to identify a plausible cause for it. Quartile coefficients of dispersion (QCD) of growth rate data were calculated at different levels of subdivision . Their distributions show that differences of growth rate between assays (isolate level of data subdivision) contribute the most to data dispersion: while 0.76% of the dishes data and of 7.1% of the assays data have a QCD larger than 0.125, data of 38 heterokaryons (26 %) have a QCD larger than this value. A QCD larger than 0.125 was subsequently considered the hallmark of high level of data dispersion throughout this study. Data dispersion between assays in the parental homokaryons is weakly correlated to a lower median growth rate _2 = 3,25 mm/day; median32(9)_2 = 7,125 mm/day; effect size r = 0.8589; P < 0.001; two tailed]. This data subset was used to show that 9(32) has indeed the slowest growth rate of all heterokaryons formed with Ho9 as acceptor parent, pointing out the fact that its combination with Ho32 is unique in this sample , , and , for three reasons: (1) differences of phenotype of their heterokaryons are statistically grounded; (2) the complete offspring resulting from reciprocal crossings of their three parents is available for a grouped analysis; (3) based on results of previous SNP call and filtering, mitochondrial haplotypes of parents Ho9 and Ho18 were supposed to be identical, which was expected to facilitate genotyping. Using data of the assay with the highest median of each heterokaryon and each parent of these three pairs, a series of 36 statistical pairwise tests was carried out, in order to assess differences of growth rate  grows significantly slower than any other heterokaryon made with Ho9 as acceptor parent  and 18(9) is significant too. Other filtering of the SNPs called from nuclear and mitochondrial genome sequences of all homokaryotic isolates were set in order to identify markers linked to these two loci.Phenotypic variance between the six isolates of pairs , , , and their three parents are best explained by the intervention of different alleles at a minimum of four nuclear loci, and mne3 . Specifir parent  shows thnc-ORF5 in the mitochondrial genome sequence of H. irregulare . In H. irregulare, nc-ORF5 is predicted to encode a protein of unknown function and made of 311 amino acid residues, annotated in the mitochondrial proteome of H. irregulare under NCBI accession YP009048485.1. Transcript and coding sequence of this locus were found to be shorter than predicted in the 5' end . Interestingly, a second indel identified after mitochondrial SNP filtering is localized 44 bp downstream of the first SNP, in the promoter/5' UTR sequence of the same ORF . Ho9 and Ho18 have the same allele of this indel. Overall, three different alleles of nc-ORF5 promoter/5' UTR exist in H. parviporum homokaryons used in this study, depending on the allelic combination of these two indels: Ho18 and Ho32 have a first allele mt2-1, Ho9, Ho11, Ho20, Ho26, Ho27, Ho30, and Ho32 have a second allele mt2-2, and Ho2, Ho5, and Ho24 have a third one mt2-3 .- for mne2, a partially negative interaction between mitochondrial allele nuc2-1 for sure  with the following mutation events, all in exon 5  with a single mutation due to a nucleotide transition C to G in exon 2 . One SNP was found 60 bp downstream of the stop codon of the gene encoding the hypothetical calcium uniporter already mentioned (H. irregulare accession number: XM_009552795.1). One SNP was found 113 bp upstream of the start codon of a gene encoding a beta subunit a of putative F1F0-type ATPase (H. irregulare accession number: XM_009544597.1). One SNP was found 11 bp upstream of the start codon of a gene encoding another mitochondrial carrier protein (H. irregulare accession number: XM_009545544.1).Of the 793 SNPs not localized into an exon of a Our phenotypic data show that two major competing trends are at work in heterokaryons, with opposite effects on growth rate. The first trend is “heterokaryon vigor”  after carrying out different growth rate assays over time. These heterokaryons can nevertheless grow as fast as the others. Their genetic potential for this specific trait is therefore not different from the rest of the heterokaryons. The fact that their phenotype does vary more between assays is due to other factors, unidentified so far, although probably linked to the heterokaryotic state, during which parental haploid nuclei coexist in the same cytoplasm, but do not fuse Raper, . The hetH. parviporum  and a genomic locus (nuc2) involved in a genetic interaction in trans across the two genomes. Different combination of alleles at these two loci contribute to phenotypic variation in the offspring of the crossings with these three isolates.Analysis of heterokaryons growth rate data showed that higher values reflect their genetic potential for this trait better, which made possible the identification of pairs of heterokaryons issued from the same parents, but whose phenotypes are different. It confirms our previous detection of mitochondrial genetic effects in the same cohort of heterokaryons of nuc2 were searched using phenotypic information of heterokaryons from all other crossings involving parent Ho9 and SNPs markers called from the genome sequences of all homokaryotic isolates , but also carrying mutations that might affect their expression level are considered the best possible candidates at this stage. Both are involved in molecular trafficking to the mitochondrion . Although the gene encoding a calcium uniporter cannot be ruled out at this stage, the other one encoding a mitochondrial carrier protein is a more likely candidate for nuc2 for several reasons: Mutations in this gene result from multiple transversions (3) and one indel in the same exon, whereas the mutation in the gene encoding the calcium uniporter results from a single transition; A multiple protein alignment with orthologs from other species shows that substitution R to K in the sequence of the carrier is in a conserved motif, and this substitution is present in Ho9 and Ho32 only; Amino-acid substitutions/deletion observed in the carrier are more likely to lead to a change of balance between structure and function.Candidates for genomic locus nuc2, but a non-conserved gene instead, with orthologs in the Russulales order only. The ortholog of this gene in the mitochondrial genome of H. irregulare has been previously identified as nc-ORF5  and by excluding proteins from the Russulales retrieved exclusively a hypothetical protein from soilborne gammaproteobacteria , and could be used for that purpose. Another option would be to shuffle mitochondria into various homokaryotic nuclear backgrounds by reisolating homokaryotic conidia from heterokaryons cultures  studying the expression of all mitochondrial and nuclear candidate genes to confirm or infirm hypotheses made about them, (2) providing molecular genetics evidence of mitonuclear interaction by carrying complementation experiments of homokaryon Ho9 with wild-type copies of candidate nuclear loci for Heterobasidion spp. as possibly involved in virulence variation related to mitochondrial background  can be found in the article/P-HC and ÅO: conceived and designed the experiments, performed the experiments, analyzed the data, and revised the paper. P-HC: exploratory data analysis, statistics, and bioinformatics and wrote the paper. ÅO: additional growth rate assays. All authors contributed to the article and approved the submitted version.This work was supported by the Carl Tryggers Foundation for Scientific Research.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Conventional selection criteria are not consistently able to discriminate between responders and non-responders to cardiac resynchronization therapy (CRT). The objective of this study was to evaluate the usefulness of quantitative gated single-photon emission computed tomography (SPECT) in predicting the response to CRT. This prospective cross-sectional study included 25 patients with advanced congestive heart failure who underwent quantitative gated SPECT before and after CRT implantation. Patients with the left ventricular (LV) lead positioned at the latest activation segment away from the scar had a significantly higher chance of responding than those with the lead positioned at a different area. Responders were likely to have a phase standard deviation (PSD) value of >33°, with 86.6% sensitivity and 90% specificity, and a phase histogram bandwidth (PHB) value of >153°, with 100% sensitivity and 80% specificity. Quantitative gated SPECT can help refine patient selection for CRT implantation, using PSD and PHB cutoff points, in addition to guiding the positioning of the LV lead. Phase analysis using gated SPECT was recently evaluated for the assessment of LV dyssynchrony, and a quantitative gated SPECT (QGS) algorithm (from Cedars-Sinai) has been used to provide quantitative parameters for the assessment of LV mechanical dyssynchrony.This study sought to explore the clinical value of QGS in predicting the response to CRT.The present study included 30 patients with advanced congestive heart failure who were eligible for CRT implantation according to the European Society of Cardiology guidelines of pacing released in 2013; patients with symptomatic heart failure despite optimal pharmacological therapy, New York Heart Association (NYHA) functional class III, and ambulatory class IV; and patients with a left ventricular ejection fraction (LVEF) of ≤35%, sinus rhythm, and a QRS duration of ≥120 ms for left bundle branch block (LBBB) morphology or ≥150 ms for non-LBBB morphology.Patients with a life expectancy of <1 year, pregnant women, patients with atrial fibrillation, and patients with previously implanted pacemakers were excluded.This study was approved by the Ain Shams University ethical committee and was performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. Informed consent was obtained from all patients.Data collection was completed for all patients, including history-taking with an emphasis on age, sex, risk factors , and a history of previous events (including previous acute coronary syndromes or coronary angiography). Patients were asked about heart failure symptoms, their functional class according to the NYHA classification, and history of current heart failure medications. A questionnaire was also given to patients to examine their quality of life (QOL) using the Arabic version of the Minnesota Living with Heart Failure questionnaire (MLHFQ). The QRS duration was calculated for all patients using 12-lead electrocardiography (ECG), and the QRS morphology was recorded.All eligible patients underwent resting ECG gated SPECT with technetium (Tc)-99m sestamibi at baseline prior to CRT implantation and at 6 months post-CRT implantation to determine global systolic synchrony parameters, the segment with latest mechanical activation, the extent of perfusion defect, and scar burden; the estimation of LVEF and LV volumes (end-diastolic volume [LVEDV] and end-systolic volume [LVESV]) was also carried out.3The resting images were acquired 1 h after intravenous injection of 20–30 mCi of Tc-99m sestamibi using a dual-headed cardiac dedicated gamma cameral system  equipped with a low-energy high-resolution parallel hole collimator operating in continuous rotation around 360° according to the local protocol of the nuclear cardiology laboratory. An ECG R-wave detector was used to provide gating to acquire 8 emission frames per cardiac cycle.7 The presence of a large total perfusion defect encompassing ≥30% of the LV was diagnosed as ischemic cardiomyopathy.8Visual and semiquantitative analyses were used to obtain a score to represent perfusion for each of the 17 segments of the myocardium based on 3 short-axis slices and a representative long-axis slice to depict the apex. Each segment was scored according to the degree of radiotracer uptake. The defect size was expressed as a percentage of the LV, as previously validated in a phantom model.Automated computer-generated LV volumes and LVEF were obtained from short-axis slices of electrocardiographically gated rest myocardial perfusion SPECT datasets using the AutoQUANT (QGS) software program .3Reconstructed images were analyzed using a modified version of the QGS algorithm  that provides quantitative parameters for the assessment of LV dyssynchrony. In the present study, a histogram and polar map were constructed from the phase value for the entire LV. The phase standard deviation (PSD) and phase histogram bandwidth (PHB) were calculated and examined as the global dyssynchrony parameters, and the segment with the latest mechanical activation was identified..9All patients underwent CRT implantation, and all leads were implanted transvenously by physicians who were blinded to the results of the SPECT examination. No intraoperative hemodynamic evaluation was performed. The pacing leads were connected to a CRT implantable device . Then, after CRT implantation, biplane fluoroscopy was performed using orthogonal views (left anterior oblique at 60° and right anterior oblique at 30°). The images were analyzed to determine the anatomic location of the LV lead using the 6-segment model The LV lead position was determined using fluoroscopy and classified retrospectively according to the results of the SPECT analysis to be concordant or discordant with the site of the most-delayed mechanical activation determined prior to implantation. The lead was considered concordant when it was found to be adjacent to the latest contracting segment of the LV assessed using SPECT. On the other hand, the LV lead was considered discordant if it was placed in any other coronary sinus tributary that was not adjacent to the latest contracting LV segment.Patients were assessed 6 months after CRT implantation for the following: NYHA functional class; QOL, using a questionnaire according to the MLHFQ; and QGS to determine LV volumes, LVEF, and global dyssynchrony indices to define the response to CRT.10Responders were defined as patients with a ≥5% improvement in LVEF as well as an improvement of ≥1 NYHA class, whereas non-responders were defined as patients who showed no improvement or a <5% improvement in LVEF and no improvement in NYHA class.t test when the data were parametric or the Mann–Whitney test when the data were non-parametric, and comparisons between paired data were performed using a paired t test. Pearson and Spearman correlation coefficients were used to assess the relationships between 2 parameters in the same group. Logistic regression analysis was used to assess predictors of CRT response after implantation.Data were coded and entered into SPSS version 22  for analysis. Qualitative data were presented as numbers and percentages. The chi-squared test was used to compare qualitative data, while Fisher’s exact test was only used when the expected count in any cell was <5. Quantitative data are presented as means, standard deviations, and ranges. Comparisons between 2 groups with quantitative data were performed using an independent P ≤ .05 was considered statistically significant.Receiver-operating characteristic (ROC) curves were constructed to determine cutoff values for PSD and PHB. The confidence interval was set to 95%, and the margin of error accepted was set to 5%. Table 1. The mean MLHFQ score was 68 ± 15 points and 68% of cases were NYHA class III, whereas 32% were NYHA class IV. The mean QRS duration was 156 ± 12 ms with 56% of cases having an LBBB morphology. The mean echocardiographic-derived LVEF was 21.6% ± 5.2%.Thirty patients were enrolled in this study; however, 2 patients died and contact was lost with 3 patients. Therefore, a total of 25 patients fulfilled the inclusion criteria and completed the 6-month follow-up assessments. The mean age was 49 ± 9 years, with 56% of the population being male and 44% of cases having an ischemic etiology. The distribution of risk factors is shown in . ECG findings revealed that the incidence of LBBB was significantly higher in the responder group, whereas the QRS duration showed no significant difference between the 2 groups.These 25 patients were subdivided according to the criteria for CRT response into 2 groups: responders (n = 15) and non-responders (n = 10). Both groups were comparable in terms of demographic data and risk factor distribution. The assessment of symptoms and QOL revealed that the responders showed significant degrees of NYHA class III and IV compared to the non-responders, whereas there were no significant differences in QOL .A comparison of the myocardial perfusion data of both groups showed that the responder group had significantly smaller LV volumes (LVEDV and LVESV) at baseline. The responder group also presented a greater baseline degree of mechanical dyssynchrony as determined using phase analysis. On the other hand, patients in the responder group had a significantly smaller perfusion defect. In the responder group, there was also a significantly higher percentage of patients with coronary sinus lead position concordant with the area of latest activation and not adjacent to scar tissue P < .001). Considering SPECT parameters, the responder group showed significant improvements in the dyssynchrony indices (P < .001) and significant reverse remodeling with improvements in LVEF .At the 6-month follow-up, both groups were compared using MLHFQ scores and SPECT parameters. The responder group showed significant improvements in the MLHFQ score compared to the non-responder group  negatively correlated with the change in LVEF. This implies that a lower MLHFQ score (better QOL) correlates with improved LVEF, and the lower the dyssynchrony indices (better synchrony), the greater the improvement in LVEF. On the other hand, the baseline QRS duration was not significantly correlated with the change in LVEF .In the present study, univariate regression analysis for predictors of CRT response revealed that non-ischemic etiology and baseline NYHA (III rather than IV) are predictors of such. Baseline SPECT-derived parameters that predict CRT response include smaller baseline LV volumes (LVEDV and LVESV), higher dyssynchrony indices (PSD and PHB), lower total perfusion defects, and placing the LV lead adjacent to the SPECT-determined latest activation and away from the scar .In the present study, ROC analysis was performed to determine the cutoff value of dyssynchrony indices to predict CRT response. A cutoff value for PSD of 33°, with a sensitivity of 86.6% and specificity of 90% , was determined. Therefore, patients with PSD > 33° were likely to be responders. A cutoff value for PHB of 153°, with a sensitivity of 100% and a specificity of 80% , was also determined, and patients with PHB values > 153° were likely to be responders 11 Current guidelines for biventricular pacing adopt the electrographic criteria in terms of QRS duration and morphology that reflect electrical rather than mechanical dyssynchrony. However, based on these criteria, 30% of patients undergoing CRT implantation are non-responders. Efforts have been made to decrease non-responder rates, including improving patient selection, optimizing the correction of electrical and mechanical dyssynchrony, and improving postimplant care.12 Echocardiographic techniques, such as tissue Doppler and strain rate imaging, have been used to identify mechanical dyssynchrony. However, these methods have some disadvantages as they lack reproducibility and the ability to differentiate between responders and non-responders to CRT.13An important aspect in the management of heart failure is to obtain and maintain optimal electrical activation of the cardiac chambers as well as atrioventricular synchrony.3 Thus, using reproducible SPECT dyssynchrony indices to address the mechanical dyssynchrony has been suggested to refine the criteria for CRT response. The assessment of LV mechanical dyssynchrony using phase analysis of gated myocardial perfusion SPECT was feasible in all patients using 2 selected indices—namely, PSD and PHB. As CRT is indicated to synchronize ventricular pacing, CRT will fail to produce tangible results if the baseline dyssynchrony is below a certain threshold. We explored the potential of baseline dyssynchrony indices to predict the response to CRT. In the present study, a cutoff point of 33° for PSD was detected, with a sensitivity of 86.6% and specificity of 90%, whereas a cutoff point for PHB of 153° was detected, with a sensitivity of 100% and specificity of 80%. These results are close to those from Azizian et al., who reported cutoff points of 21° for PSD and 112° for PHB, respectively.14 In addition, it was demonstrated that the improvement in mechanical dyssynchrony indices significantly correlated with the improvement in LVEF, a result which is quite similar to that highlighted in the VISION-CRT study.15SPECT has been used to determine dyssynchrony indices using phase analysis, which has been shown to be reproducible and has the potential to predict the response to CRT.15 Prior to CRT, it seems logical to select the position of the LV lead insertion in the coronary sinus vein as one adjacent to the latest electromechanical activation–viable segment to achieve optimal resynchronization.16 The priority is to achieve a stable LV lead position with a suitable threshold in the absence of diaphragmatic pacing in the posterolateral area. All LV leads were positioned in the mid-LV segment in the right anterior oblique projection to avoid apical insertion of the LV lead, as the MADIT-CRT study showed that the apical position is less effective and even harmful, increasing the risk of death.17 In the present study, positioning the LV lead concordant with the latest activation segment was a predictor of CRT response. These results agree with those concluded in the study by Boogers et al. using SPECT and similarly in the TARGET trial with speckle-tracking site analysis.19 Nevertheless, in the VISION-CRT study using SPECT, the investigators failed to show this association that was possibly attributed to a lack of operator blinding to the echo information of sites of scar that could have influenced the lead positioning decision.15 It is also noteworthy that reliable echocardiographic measurements require expertise in order to obtain consistent and reproducible results.SPECT imaging is not only capable of dyssynchrony evaluation but also allows simultaneous assessment of LV perfusion and function. In addition, it can assess myocardial scar location and severity for optimizing CRT in patients with heart failure.21 Additionally, patients with non-ischemic cardiomyopathy are more likely to be responders than those with ischemic cardiomyopathy.22 In the present study, the total defect size that reflects scar burden was significantly larger in the non-responder group. This finding is supported by Adelstein et al. and Sciagrà et al., who reported that patients with extensive resting perfusion defects had a limited clinical response to CRT.23 This may be explained by the direct correlation between LV scar and LV dyssynchrony. Samad et al. used SPECT imaging to show that the presence of LV scar and its extent were independent predictors of mechanical dyssynchrony.24 In addition, Ludwig et al. proposed that PSD values were increased by the presence of scar tissue.25Extensive scar tissue in the LV may hinder the response to CRT. Accordingly, patients with extensive scarring in the LV were less likely to be responders to CRT.27In the present study, the relationship between the scar and LV lead position had a significant impact on CRT response. Within the responder group, only 27% of patients had the LV lead concordant to the scarred area, while 80% of patients in the non-responder group had the lead concordant to the scarred area. This result was similar to the findings reported by Leyva et al. and Bleeker et al., who found that placing the LV lead away from the scar tissue gave clinically better results after implantation of CRT. This could be due to the fact that scar tissue could not respond or contract compared to non-scar tissue; therefore, placing the LV lead adjacent to scar tissue would result in an impaired response.30 In addition, a lower response in patients with NYHA class IV is similar to that reported by De Sisti et al., which could be attributed to the more adverse remodeling and higher baseline dyssynchrony in patients with NYHA class IV.31 The presence of LBBB morphology has been shown to be significantly higher in the responder group, which is commensurate with the guideline recommendations. In the present study, responders showed a significantly smaller heart (lower EDV and ESV) at baseline, which is supported by the findings reported by Azizian et al., who found that responders showed less dilatation in the LV than non-responders, although the difference was not statistically significant.14 In addition, significant reverse remodeling was observed in the responder group after CRT implantation compared to the non-responder group, consistent with the results of the study by Boogers et al.18It is noteworthy that other factors that influence the CRT response were demonstrated in the study. The responder group had a significantly higher proportion of female patients, NYHA III (rather than IV), non-ischemic etiology, LBBB morphology, and baseline smaller LV volumes. The higher female sex prevalence in the present study is consistent with the findings reported by Cheng et al., which is possibly explained by the greater rates of non-ischemic etiology and deaths from pump failure rather than sudden cardiac death in female patients.This study has some limitations that need to be reinforced by more specific larger studies in order to contribute to clinical practice. One of the limitations is the unfeasible application of multivariate analysis due to the small number of patients enrolled.In conclusion, SPECT represents a “one-stop shop” non-invasive imaging modality that has the potential to refine patient selection for CRT implantation and predict and optimize the CRT response. It not only enables assessment of mechanical dyssynchrony with reproducible indices but also allows for simultaneous assessment of LV perfusion and function. In addition, it can assess myocardial scar location and severity and guide placing of the LV lead adjacent to the gated SPECT-derived latest activation segment and away from the scarred area to achieve a better response.


Neurocognitive disorders due to human immunodeficiency virus (HIV) remain highly prevalent, specifically mild forms despite effective antiretroviral therapy (ART). Dolutegravir-based regimens are the first line of treatment for adult HIV-positive patients. Controversies exist regarding the neurocognitive effects of dolutegravir. Evidence regarding the neurocognitive effects of dolutegravir is important, in support of its use in patients with HIV-associated neurocognitive disorders (HAND).This study aimed to describe the change in cognitive function using the International HIV Dementia Scale (IHDS) and Brief Neuropsychological Cognitive Examination (BNCE) in HIV positive, treatment naïve patients before and 3 months after initiation of ART using a dolutegravir-based regimen.The HIV initiation clinic of Hillbrow Community Health Centre in Johannesburg.This prospective, quantitative cohort study assessed adult HIV-positive patients who were ART naïve being initiated on a dolutegravir-based regimen, using the BNCE and IHDS at baseline and after 3 months of treatment.Z = 1.84, p = 0.033) and time to complete BNCE . BNCE total results showed improvements that were not significant ; however, Part 2 of the BNCE reflecting that of executive function showed significant improvements .Neurocognitive test results of 26 participants showed significant improvements for IHDS (The trend of neurocognitive function is towards improvement in HIV-positive treatment naïve patients who receive 3 months of dolutegravir-based ART.The findings support the use of dolutegravir-based regimens in the treatment of patients with HIV-associated neurocognitive disorders.HIV-associated neurocognitive disorders; BNCE; IHDS; dolutegravir; neurocognitive screening; neurocognitive impairment; South Africa. It was hypothesised that results would show improvement. Secondary objectives included to compare patient sociodemographic data  as well as blood results, specifically baseline CD4 count and viral load (VL) measured at 3 months post-ART initiation and correlate these findings to change in neurocognitive results.This study was a prospective, quantitative and descriptive cohort study. It was conducted between June 2021 and July 2022 at the HIV initiation clinic at Hillbrow Community Health Centre in Johannesburg. This unit falls under the district level of care in the public health sector of the Gauteng Department of Health in South Africa.The study sample population comprised English-speaking and consenting, adult HIV-positive patients who were ART naive being initiated on a dolutegravir-containing regimen. Exclusion criteria consisted of any previously diagnosed neurocognitive disorder or the presence of other known causes of neurocognitive impairment such as depression, stroke, intellectual disability, traumatic brain injury and current or recent substance use. Patients older than 60 years were excluded from the study to preclude age-appropriate neurocognitive decline as a confounding factor. The calculated required sample size was 33 participants to achieve significant results with a certainty of 95%. The intended sample size was 50 participants to account for participant dropout and other unforeseen circumstances. Patients attending the HIV initiation clinic were chosen by stratified sampling. Adult patients who were ART naïve and to start a dolutegravir-containing regimen were given information regarding the study by use of a pamphlet and provided written consent to participate in various aspects of the study. A screening questionnaire was then provided comprising inclusion and exclusion criteria. Eligible participants were allocated a participant number and included in the study.5 The maximum score is 12 with scores of 11 or 12 considered normal and scores of 10 or lower indicating need for further neuropsychological testing. The second test, the BNCE, is made up of two parts with five subtests in each part. Part 1 subtests are mainly related to conventional types of information processing  and Part 2 subtests are aimed at assessing the performance of executive functioning.18 Specific to the South African population, modification of Part 1, section B of the BNCE titled presidential memory was required, in order to be appropriate to the knowledge and culture of the study group. Questions were adapted from assessing presidential memory of the United States to that of African presidential history that is well known to the people of South Africa. This maintains a culturally fair and appropriate measure of long-term memory and crystalised intelligence, in the absence of other culturally appropriate tests. The maximum score is 30 with total scores of 27 or less indicating cognitive impairment. Scores are then further divided into mild (22–27), moderate (10–21) and severe (0–9).18 The time taken to complete the BNCE was also considered in evaluating cognitive improvement. This is not a requirement for the administration of the BNCE, however, is useful when assessing processing speed in the consultation.18 Neurocognitive testing was conducted on the day of ART initiation and repeated after approximately 3 months after confirming treatment adherence. Blood results were documented, specifically CD4 count at baseline and VL after 3 months of receiving ART.The primary investigator collected and documented demographic information and assessed participants using the IHDS and BNCE. The IHDS screening tool is a three-part examiner administered test comprising of a non-dominant finger-tapping test, a non-dominant Luria hand sequence and a four-word recall test.www.R- project.org).Of the 63 participants that completed the screening questionnaire, data from a total of 26 participants were used in the analyses. These participants completed the 3-month follow-up to produce useable data to address the aims of the study. The data were collated in Microsoft Excel™ and statistically analysed in R software , logistic regressions for dichotomous outcomes, general linear regressions and Wilcoxon matched pairs tests . Some analyses were one-tailed based on directional hypotheses (e.g. Wilcoxon tests), and all other analyses were two-tailed. Model alpha was set at 0.05. Data are reported as counts and percentages  or mean and standard deviation (continuous data) and presented descriptively in the text, tables and figures.19 Approval was also granted by The National Health Research Database (Ref: GP_202008_151). Written consent was obtained from all individual participants involved in the study. Confidentiality was maintained by the removal of identifiable information and use of participant numbers.Ethics clearance was obtained from the University of Witwatersrand Human Research Ethics Committee (Clearance Certificate No. M201102). All procedures performed were in accordance with the 1964 Helsinki Declaration and it’s later amendments.Of the 63 patients screened for participation, 38 were eligible. Excluded participants comprised mostly males and the majority due to substance use. After a loss to follow up drop out rate of 31, 6%, 26 participants completed the study . The day2 = 9.85, df = 1, p = 0.002) and all were black African ethnicity. The years of formal education ranged from 7 to 14 years with a mean of 11.0 (s.d. = 1.7) years. CD4 counts were found to have a mean of 304.1 (s.d. = 228.2) cells/mm3 and when categorised into below or above counts of 200 cells/mm3, the difference was not significant . The viral load measured at 3 months varied with significantly more patients showing virological suppression at levels of 0–20  (The demographic data as depicted in = 0.032) .Z = 1.84, p = 0.033)   (The neurocognitive tests at baseline and at 3 months showed that IHDS results increased significantly from a baseline mean of 8.3 (s.d. = 1.6) to a 3 month mean of 9.1 (s.d. = 2.1) (Wilcoxon test: = 0.033) . The BNC= 0.075) . In cont= 0.007) .Z = 297.5, p < 0.001) and BNCE 2    . At base< 0.001) . When co= 0.043) .p = 0.009). The BNCE score at baseline also decreased with every year of increasing age; however, this was not found to be significant (p = 0.065). Years of education were found to be a significant predictor of both IHDS and time taken to complete BNCE. A one year increase in years of education increased the IHDS 1 and 2 scores significantly . Similarly, with every 1 year increase in years of education, the time taken to complete the BNCE decreased both at baseline and 3 months later . While BNCE total scores at baseline and 3 months were also found to increase with every year of education, this was not found to be significant . The correlation between socio-demographic variables and improvement in neurocognitive test scores was not found to be significant  and decrease in time taken to complete the BNCE (p = 0.015). In comparison, those with CD4 counts of > 200 cells/mm3 showed no significant change in IHDS, BNCE and time scores  (Participants with a baseline CD4 count of < 200 cells/mmctively) .No significant correlation was found between VL measured at 3 months and improvement in neurocognitive test results .20 The authors concluded that while 6 months was long enough to see VL suppression in both plasma and CSF, this may not be enough time to confirm change in cognitive function.20 The time factor may explain the findings in that not enough time was allowed for all aspects of cognition to improve between testing. Cysique et al. found that cognitive improvement can start as early as 12 weeks; however, it occurs more often between 24 and 48 weeks.3 In order to detect clinically appreciable differences, a follow-up of longer than 2 years is suggested.21 Another factor to consider is the time to improvement of specific domains of cognitive function. It is well known that HIV primarily impacts functions of subcortical and striatal brain regions, namely executive function, motor and information processing speeds and memory recall.5 The IHDS is limited to the evaluation of motor speed, memory recall and psychomotor functions.22 In contrast, the BNCE covers a broader range of cognitive functions with a specific focus on executive function.17 It is not clear which aspects of cognitive function are first to improve with ART with some short-term studies showing improvement in all domains except motor processing speed.23 This is an area requiring further research. Another explanation for the findings could be that high rates of persistent milder forms of neurocognitive impairment are commonly seen despite adequate ART with the BNCE being more appropriate in its recognition.24This study aimed to describe the changes in cognitive function at baseline and 3 months after initiation of ART using a dolutegravir-based regimen with the hypothesis that results would show improvement. Significant improvements were noted in both IHDS scores and time taken to complete the BNCE. This supports improvement in subcortical neurocognitive function tested in the IHDS, as well as processing speed indicated by the time taken to complete the BNCE. While the total BNCE scores did show improvements from baseline, these results were not found to be significant. This is consistent with findings from a study done in 2020 assessing change in cognitive function over a 6 month period of using dolutegravir as assessed by the Montreal Cognitive Assessment (MoCA).18 In assessment of HAND, one can expect Part 1 scores to be relatively preserved and Part 2 scores to show deterioration, with the difference being more pronounced in severe disease.18 Results confirmed this in that majority of participants had lower scores in Part 2, confirming the subcortical origin of pathology as well as emphasising the burden of mild neurocognitive impairment based on point discrepancies found between the two parts. In moderate to severe dementia, the score in Part 2 shows earlier and quicker deterioration and with disease progression is expected to remain consistently lower than Part 1 scores.18 Interestingly, the results show improvement of BNCE part 2 scores at 3 months and a lower percentage showing the larger discrepancy between the two parts. This trend is in keeping with the subcortical origin of HAND and the quicker deterioration and therefore improvement with treatment in these areas of cognition. Results also show more improvement in Part 2 than Part 1 scores. This confirms that significant improvement in certain areas of cognitive functioning is taking place over the short period examined. However, this does not equate to overall improvement in cognitive functioning.The BNCE’s advantage over other screening tests is its ability to assess both conventional, frequently used cognitive functioning as well as more advanced levels of executive functioning allowing for detection of milder forms of cognitive impairment.25 This is considered as having high CNS penetration.25 While higher CPE scores are associated with improved control of viral replication in the CNS, its correlation to neurocognitive impairment remains controversial with ongoing concern of neurotoxicity.25 Despite this possibility, a recent study concluded that ART with higher CPE is associated with modest improvement in global neurocognitive functioning, with less improvement noted in specific cognitive domains such as attention, working memory and learning.26 This could explain why significant improvements were noted in the IHDS and processing speed and not in BNCE with its strong emphasis on higher orders of cognitive function. Overall, it has been found that higher CPE regimens are slightly more likely to preserve or improve cognitive function than regimens with lower CPE scores.26 Research comparing dolutegravir-based regimens with other regimens of lower total CPE value would be beneficial.With the focus of this study on dolutegravir, which when combined in the currently recommended regimen, contributes four points to a total CPE score of seven.26 It occurs in 4% – 20% of patients treated with ART, with some experiencing ongoing neurological symptoms.26 This could explain the findings of lack of correlation between neurocognitive improvement and serum VL suppression.Cerebrospinal fluid viral escape should also be considered in assessing effects of CPE on neurocognitive function. This occurs when plasma viral suppression is achieved, while CSF viral replication continues.21 Evidence also shows that age-related cognitive decline is exacerbated by HIV.10 Although the study excluded participants over the age of 60 to account for age-related decline in cognition, some elements of executive function have been found to start declining as early as 45 years of age.27 This could also explain why we see a correlation between increasing age and BNCE results and we do not see the same correlation with IHDS results, with its limited assessment of executive function. Another aspect to consider with increasing age is that drug pharmacokinetics may be less effective, negatively impacting the relationship between CPE score and neurocognitive improvement.26As expected, cognitive test results of BNCE were lower with increasing age. In the ageing HIV population, both accelerated and natural decline can be seen due to multiple factors, with AIDS as the predominant cause and hence expectation is to see a negative effect of age on cognitive function despite adequate treatment.27 This study confirmed that for each year increase in years of education, there was an increase in neurocognitive score as well as faster processing speed.The cognitive reserve hypothesis holds that factors such as higher levels of education, socioeconomic status and baseline intelligence can be protective against clinical manifestations of brain pathology.8 This study did not find any correlation between gender and neurocognitive function; however, this could be affected by over-representation of female participants.Research regarding gender as a risk factor for neurocognitive impairment is conflicting.28 This study confirms that those with lower baseline CD4 counts were more likely to show improved neurocognitive scores and processing speed 3 months after receiving treatment. This could be accounted for by the association of baseline CD4 cell counts of less than 200 cells/mm3 with increased proliferation of HIV within the CNS.29 Another consideration is the possibility of delirium at baseline prior to the initiation of ART. Risk factors for delirium include the presence of neurocognitive impairment as well as acute and opportunistic infections, which more commonly occur with advanced immunosuppression.30 In contrast, multiple studies have found that markers of advanced HIV such as lower CD4 counts were not a significant risk factor for delirium.30 The results support early initiation of ART to assist with various degrees of neurocognitive impairment.Many large studies have confirmed that a low nadir CD4 is an indicator of neurocognitive impairment as well as a predictor for future diagnosis of HAND.This study contributes towards the growing evidence regarding effects of ART on neurocognitive function as well as the need for more evidence around the use of dolutegravir.31 These factors increase the false-positive rate of HAND diagnosis, questioning the validity of the current criteria with a move towards more emphasis on clinical diagnosis.31Limitations included a small sample size with a significant female predominance, which may have impacted on some results noted. Additionally, the BNCE required adjustments to make the test culturally appropriate. While this is the approach taken in practice and necessary to ensure the suitability of the test to South Africans, construct validity of these changes has not been established. Furthermore, a control group for comparison would have been useful to account for practice effects when interpreting improvements in results. This is an area for further research. It is also acknowledged that complex multifactorial factors contribute towards low cognitive test performance, including socioeconomics, quality of education and comorbidities.Based on the findings, one can conclude that the trend of neurocognitive function is towards improvement in HIV positive, treatment naïve patients who receive 3 months of dolutegravir-based ART. While the dolutegravir cannot be implicated as the main factor contributing towards improvement, the findings support the use of dolutegravir-based regimens in the treatment of patients with HIV-associated neurocognitive impairment. Future research should aim at assessing patients over a longer period to confirm clinically significant improvements. Comparison to other regimens would also be beneficial.


This secondary analysis examines the self-rated aspects of health-related quality of life, including physical and psychological well-being, social and peer support, and coping at school, among healthy children who were enrolled in a sleep manipulation trial. Does losing sleep affect health-related quality of life in children?In this secondary analysis of a randomized crossover trial involving 100 healthy children aged 8 to 12 years, receiving 39 minutes less sleep per night resulted in significantly lower physical and overall well-being, ability to cope well at school, and total health-related quality of life, especially in children with at least a 30-minute difference in sleep.Findings of this secondary analysis of a randomized clinical trial indicate that ensuring children receive sufficient good-quality sleep is an important child health issue. Little is known regarding the effect of poor sleep on health-related quality of life (HRQOL) in healthy children.To determine the effect of induced mild sleep deprivation on HRQOL in children without major sleep issues.This prespecified secondary analysis focused on HRQOL, a secondary outcome of the Daily Rest, Eating, and Activity Monitoring (DREAM) randomized crossover trial of children who underwent alternating weeks of sleep restriction and sleep extension and a 1-week washout in between. The DREAM trial intervention was administered at participants’ homes between October 2018 and March 2020. Participants were 100 children aged 8 to 12 years who lived in Dunedin, New Zealand; had no underlying medical conditions; and had parent- or guardian-reported normal sleep (8-11 hours/night). Data were analyzed between July 4 and September 1, 2022.Bedtimes were manipulated to be 1 hour later (sleep restriction) and 1 hour earlier (sleep extension) than usual for 1 week each. Wake times were unchanged.All outcome measures were assessed during both intervention weeks. Sleep timing and duration were assessed using 7-night actigraphy. Children and parents rated the child’s sleep disturbances (night) and impairment (day) using the 8-item Pediatric Sleep Disturbance and 8-item Sleep-Related Impairment scales of the Patient-Reported Outcomes Measurement Information System questionnaire. Child-reported HRQOL was assessed using the 27-item KIDSCREEN questionnaire with 5 subscale scores and a total score. Both questionnaires assessed the past 7 days at the end of each intervention week. Data were presented as mean differences and 95% CIs between the sleep restriction and extension weeks and were analyzed using intention to treat and an a priori difference in sleep of at least 30 minutes per night.The final sample comprised 100 children . During the sleep restriction week, children went to sleep 64  minutes later, and sleep offset (wake time) was 18  minutes later, meaning that children received 39  minutes less of total sleep per night compared with the sleep extension week in which the total sleep time was 71  minutes less in the per-protocol sample analysis. Both parents and children reported significantly less sleep disturbance at night but greater sleep impairment during the day with sleep restriction. Significant standardized reductions in physical well-being , coping in a school environment , and total HRQOL score  were reported by children during sleep restriction, with an additional reduction in social and peer support  in the per-protocol sample analysis.Results of this secondary analysis of the DREAM trial indicated that even 39 minutes less of sleep per night for 1 week significantly reduced several facets of HRQOL in children. This finding shows that ensuring children receive sufficient good-quality sleep is an important child health issue.ACTRN12618001671257Australian New Zealand Clinical Trials Registry:  A 2016 systematic review6 reported an inverse association between sleep duration and HRQOL in 3 studies but rated such evidence as very low quality. Although several recent large studies have reported associations between sleep and HRQOL in children,19 these studies have all been cross-sectional and unable to determine causality. Few studies have used objective measures of sleep, which can differ markedly from measures obtained from questionnaires.21 As a recent study showed, device-measured sleep had no association with HRQOL, whereas self-reported ratings of poor sleep quantity and quality were associated with lower HRQOL.19To date, most research has examined the association of more medically related sleep issues, such as obstructive sleep apnea,22 The aim of this secondary analysis was to determine the effect of mild sleep deprivation  on HRQOL in children without major sleep issues.To our knowledge, no experimental studies have yet determined the effect of manipulating sleep on HRQOL in healthy children to the extent that might represent the levels of mild sleep deprivation that many children may experience today.23  or assent (by children) was obtained at the first visit after a verbal explanation of the protocol and an opportunity to ask any questions. We followed the Consolidated Standards of Reporting Trials (CONSORT) reporting guideline.25The DREAM randomized crossover trial investigated how mild sleep deprivation influenced eating behaviors and activity patterns in children aged 8 to 12 years in the naturalistic home environment. Detailed information on this trial is provided in the protocol23  and a pr26 Only children with parent (or guardian)-reported time in bed of 8 to 11 hours per night were included to ensure that the intervention (restriction and extension of time in bed) did not place them in the not recommended category of sleep duration for this age group .27 Parents and children were emailed separate written information sheets prior to enrolling.Healthy children were recruited by advertisement between October 2018 and March 2020 and were eligible to participate if they were aged 8 to 12 years; lived in the wider Dunedin area in New Zealand; had no underlying medical conditions or medications that could affect sleep; and scored 39 or lower, which indicated no major sleep problems, on the Sleep Disturbance Scale for Children.28 Randomization was undertaken by the research staff following baseline measures. Participants or intervention deliverers could not be blinded to the intervention group (sleep extension or sleep restriction), but those who were analyzing the accelerometry data and the biostatistician were all blinded to the intervention allocation and worked using code A or B to denote group randomization order rather than sleep extension or restriction.Children were randomized to the order in which they underwent sleep restriction and sleep extension weeks and were stratified by age group (8-10 years or 11-12 years) and sex . Randomization to order was generated by one of us  using random block lengths in Stata, version 17.0 (StataCorp LLC) in a 1:1 allocation and then was uploaded to a research management program randomization module .29 Means of usual bed and wake times, determined from a 7-day sleep diary at baseline, were calculated separately for weekdays and weekend days. Researchers discussed with parents whether these means reflected usual bed and wake times and adjusted the means if required . Wake times were kept constant to mirror daily life restrictions , and interventions were administered only during the school term.To achieve mild sleep deprivation, children were asked to go to bed 1 hour earlier than usual for 1 week (sleep extension) and 1 hour later than usual for 1 week (sleep restriction), separated by a 1-week washout to allow sufficient time for children to return to their usual sleeping habits before the next intervention week.Researchers worked with families during a single problem-solving session  to identify any barriers to changing bedtimes, such as being at a sporting activity until close to the extension bedtime, that limited the ability to complete prebed activities in a timely fashion. A suggestion to counteract this barrier could have been to prepare the dinner meal earlier that day or to complete school assignments in the morning. Families received daily personalized bedtime text reminders during each intervention week.30 Parents completed the 22-item Children’s Sleep Hygiene Scale,31 which assesses the regularity of behaviors that might support or interfere with sleep and has a score range of 1 to 6 for each item, with higher scores indicating better sleep hygiene . This information was mainly collected to provide feedback to families but was occasionally used to inform discussions about bedtimes if an individual sleep hygiene practice was uncommon. Duplicate measures of height (in centimeters) and weight (in kilograms) were collected at baseline using standard procedures,23 and body mass index z scores were calculated using the World Health Organization Child Growth Reference data.32Demographic data were obtained from the parent, including age, sex, and race and ethnicity of the child; presence of any siblings; and maternal educational level . Area-level deprivation for a family was measured with the 2018 New Zealand Index of Deprivation, an index based on the New Zealand Census of Population and Dwellings data that reflects the extent of material and social deprivation used to construct deciles from 1 (least deprived) to 10 (most deprived).34Children wore an accelerometer , which was set to 30 hz at initialization and downloaded with 15-second epochs, on their right hip 24 hours a day for 1 week to measure sleep timing (onset and offset), sleep duration , and sleep quality . Actigraphy data were analyzed using a count-scaled algorithm developed in MATLAB (MathWorks Inc) that estimated sleep onset and offset as well as awakenings that were specific to each individual for each day.23 Children and their parents each completed the 8-item Pediatric Sleep Disturbance and 8-item Sleep-Related Impairment scales of the Patient-Reported Outcomes Measurement Information System (PROMIS) questionnaire.35 The responses provided a subjective assessment of the difficulties the child had in falling and staying asleep (disturbance) and daytime sleepiness and the effects on functioning (impairment). Questionnaire items referred to occurrences over the past week and used 5 frequency response options: never, almost never, sometimes, almost always, or always. The Cronbach α for the sleep disturbance scale was α = 0.87 in children and α = 0.85 in parents. The Cronbach α for the sleep impairment scale was α = 0.91 in children and α = 0.94 in parents.Actigraphy data were collected for each child for the duration of each intervention week, as occurred at baseline. Questionnaire data were collected at the end of each intervention week (day 8) during an assessment session.36 Children provided answers based on 2 response scales . The KIDSCREEN-27 questionnaire produced a total score (score range: 2.4-5.0) and 5 subscale scores: physical well-being , psychological well-being , autonomy and parental relations , social and peer support , and school environment . Children were assisted with completing the questionnaires if required; in practice, assistance was rarely needed.Children also completed the 27-item KIDSCREEN questionnaire, which assessed HRQOL over the past week, with a score range of 1 (never) to 5  or 1  to 5 (extremely), as appropriate.All analyses were undertaken in Stata, version 17.0 (StataCorp LLC). Effects of mild sleep deprivation were estimated by mixed-effects regression models, with the child as a random effect. Mean differences and 95% CIs were determined for sleep restriction compared with sleep extension. Standardized mean differences (SMDs) and 95% CIs were also calculated using a pooled SD. Residuals of models were plotted and visually assessed for homoskedasticity and normality. Complete case analyses were undertaken using the full sample (intention to treat) and were restricted to those who met the per-protocol a priori definition of a difference in sleep of at least 30 minutes per night.P < .05 indicated statistical significance. Data were analyzed between July 4 and September 1, 2022.Two-sided 37The Baseline sleep characteristics (from actigraphy) showed that children received a mean (SD) total sleep time of 8 hours and 59 minutes (45 minutes) per night and a high level of sleep efficiency , waking less than once a night on average  . BaselinAccelerometry data indicated that children went to sleep an average of 64  minutes later each night over the sleep restriction week compared with the sleep extension week, with a smaller difference (as anticipated) in sleep offset . These differences in sleep timing meant that children received 39  minutes less of total sleep time each night during the sleep restriction week. These differences were magnified in the per-protocol sample, which had a nightly difference in total sleep time of 71  minutes. As observed from the child and parent reports, more consolidated sleep was apparent, with small reductions in the number of awakenings  and wake after sleep onset  as well as a small improvement in sleep efficiency  in the full sample . By cont38 the reductions in multiple aspects of HRQOL were observed after only 1 week of less sleep. As such, we believe these findings are clinically and statistically significant and require confirmation over the longer term.Results of this secondary analysis of the DREAM trial demonstrated that even relatively small reductions in nightly sleep duration can have a considerable effect on HRQOL in children. These children received 39 minutes less sleep per night between sleep conditions over only 1 week. This loss of sleep resulted in significant reductions in the children’s physical well-being, overall well-being, and ability to cope well in a school environment. For those who achieved the a priori difference in sleep of at least 30 minutes per night, additional reductions in well-being associated with less social and peer support were also observed. While these differences may generally be considered as small but not trivial,13 appeared to involve children with clinical sleep issues, where greater benefits might be expected. In young children with obstructive sleep apnea, significant improvements in HRQOL were observed, principally in school and physical function domains, after adenotonsillectomy.9 In children with milder sleep issues , a brief clinician-delivered intervention showed short-term (3-month) improvements in psychosocial HRQOL even though the proportion of children with sleep problems had not decreased. However, benefits to HRQOL were not maintained at follow-up (12 months), and any corresponding changes in sleep duration were not reported.39 Other research that manipulated sleep in children focused more on emotional or cognitive outcomes rather than on HRQOL, reporting consistent adverse effects on mood and smaller effects on emotion and some cognitive test scores.13 We believe the findings of this trial add considerable value to the existing cross-sectional literature that shows the association of sleep duration with HRQOL in children19 because causality can now be inferred.It is difficult to compare the findings with those reported in the literature because most previous experimental studies40 We found that when children slept less, they ate substantially more calories, particularly in the evenings, all of which came from noncore foods  rather than from core foods, such as fruit and vegetables,41 which are associated with higher HRQOL.42 Children replaced this loss of sleep mostly with sedentary time and, to a lesser degree, light activity.43 Given that these activity patterns were measured using accelerometry, we do not know which specific behaviors might have changed, although anecdotally, parents reported more screen time during the sleep restriction week. In general, greater HRQOL has been associated with higher amounts of activity and lower amounts of sedentary time in children.45Previous analyses of the DREAM trial provided some insight into why these relatively modest changes in sleep might affect HRQOL, although it is difficult to disentangle which behavioral changes might influence HRQOL the most as they tend to be interrelated in children.20 However, it was interesting to observe greater sleep efficiency when sleep was restricted, suggesting more consolidation of sleep, which was supported by children and parent reports of less sleep disturbance in the PROMIS questionnaire. This finding likely reflects an increased homeostatic drive to sleep as a consequence of sleep restriction.46 It is important to note that we specifically tested sleep restriction vs sleep extension conditions to ensure the best opportunity to create a difference in true sleep, as the DREAM trial was a mechanistic study that aimed to determine the effect of mild sleep deprivation on eating and activity behaviors associated with obesity in children.24 We purposely chose to restrict sleep by a relatively small amount in an effort to mimic clinical levels of mild sleep deprivation,22 which we believed had greater applicability for public health than more severe sleep deficits often used in other sleep manipulation trials.48This study has several strengths. These strengths primarily centered on the randomized crossover trial design, wherein each child participant was able to act as their own control, with the sleep extension week providing the conditions for more sleep opportunity compared with the sleep restriction week that provided less sleep opportunity. We determined the difference in sleep on the basis of actigraphy rather than subjective measures of sleep duration, which should provide greater accuracy.23 ensuring a robust sample size and reduced risk of attrition bias. The sample was not diverse, which may limit extrapolation to other groups. As the trial was a mechanistic study, we cannot comment on the effect of sleep loss over the long term and its implications for HRQOL in children because a consistently linear association between less sleep and worse HRQOL cannot be assumed in the absence of such evidence. Furthermore, we had a single measure of HRQOL from the children; confirmatory data from different measures or parents would have been an advantage.This study also has limitations. It focused on a secondary outcome of interest, for which we did not undertake specific power calculations. Although data collection had to be concluded slightly earlier than anticipated due to COVID-19 restrictions, the total dropout rate was low at 5% compared with the projected rate of at least 20%,In this secondary analysis of the DREAM randomized crossover trial of sleep manipulation, we showed that after only 1 week of receiving 39 minutes less sleep per night between sleep conditions, children reported significantly lower HRQOL in terms of their physical and overall well-being and ability to cope well at school. These findings highlight that ensuring children receive sufficient good-quality sleep is an important child health issue.