Variabilichromis moorii from Lake Tanganyika, using samples of broods and their brood-tending parents that were collected across five field trips from the same study population. The sampled broods were either spawned during the dry season (three field trips) or during the rainy season (two trips). In all seasons, we detected substantial rates of extra-pair paternity, which were ascribed to cuckoldry by bachelor males. Paternity shares of brood-tending males were consistently higher, and the numbers of sires per brood were consistently lower, in broods that were spawned in the dry seasons compared to broods from the rainy seasons. In contrast, the strength of size-assortative pairing in our V. moorii population did not vary temporally. Seasonal fluctuations in environmental conditions, such as water turbidity, are proposed as a mechanism behind variable cuckolder pressure. Our data demonstrate the utility of long-term monitoring to improve our understanding of animal mating patterns.Mating patterns in animal populations can respond to environmental conditions and consequently vary across time. To examine this variation in nature, studies must include temporal replicates from the same population. Here, we report temporal variation in genetic parentage in the socially monogamous cichlid The online version contains supplementary material available at 10.1007/s10750-022-05042-0. Animal-mating patterns have profound effects on evolutionary processes. Mating patterns determine how an important resource—mating opportunities—is distributed among individuals in a population, often with unequal distributions between the sexes. For instance, if females are unavailable for mating for prolonged periods of time due to gestation or brood care, then it is generally expected that males will compete more intensely among one another for the few available females. The resulting competition for mates can give rise to sexual selection on traits that are positively associated with mating success, sometimes leading to the evolution of exaggerated ornaments or armaments  led to the assumption that variance in reproductive success can be roughly predicted from the social mating system. For example, social monogamy is considered to be associated with mild variance in the mating success of both sexes, since each reproductive adult will claim only one member of the opposite sex from the mating pool  horei , a maternal mouthbrooder and Variabilichromis moorii , a substrate breeder—inhabit the shallow rocky littoral of Lake Tanganyika and seasonal variation in environmental factors may be a driver of this temporal variation in brood paternity : Morley & Balshine, Amatitlania nigrofasciatum : Wisenden, Neolamprologus caudopunctatus : Schaedelin et al., Pelvicachromis taeniatus : Baldauf et al., V. moorii show positive size-assortative pairing , field trip or season, and their interaction term as predictor variables. We also calculated Pearson correlation coefficients between the male and female partner body sizes for each field trip and each season. We used a paired t-test to examine size differences between the sexes within social pairs, and a Welch’s two-sample t-test to examine body size dimorphism in the population.P = 0.0001) and were higher in the dry seasons than in the rainy seasons . Pooling data from multiple field trips by season yielded consistent results with a significant negative effect of body size  and significantly higher paternity shares of the brood-tending males in the dry season compared to the rainy season .Paternity shares of brood-tending males varied among field trips . Sire numbers per brood varied among field trips . Pairwise comparisons between field trips indicated fewer sires per brood in dry compared to rainy seasons , whereas none of the same-season comparisons revealed significant differences in sire numbers  and was higher in the rainy season compared to the dry season .The paternity shares of brood-tending males were negatively correlated with the number of additional sires that fertilized eggs in their female partners’ broods  nor did they differ among field trips .Average paternity shares of brood-tending males were 2.0 to 3.8 times higher than average paternity shares of cuckolders, and this bias was always stronger in the three dry seasons compared to the two rainy seasons . Across samples, females were on average 0.2 cm larger than their male partners , and this difference corresponds to the population-level size difference of 0.2 cm between males and females .When pooling across all five field trips, there was a significant correlation between the body sizes of male and female brood-tending partners  revealed no significant differences in paternity rates of nest-owning males between early and late spawning season , average paternity shares of guarding males increased over the course of a breeding season, ranging from less than 10% to near 100%, a pattern that is likely driven by declining occurrences of nest take-overs as the season progresses  horei  weather conditions. For instance, rainfall increases the influx of sediment into the lake, and we have repeatedly encountered poor underwater visibility during field trips early in the year  and more stable conditions of good visibility between September and November . Interestingly, the dry season ended prematurely in 2017 (Zimmermann pers. obs.), and the difference in paternity shares and sire numbers in pairwise comparisons between V. moorii, found very high rates of multiple paternity within their sample that was also taken during a rainy season. Similarly, a population of another maternal mouthbrooder from Lake Tanganyika, C. (S.) horei, experienced higher rates of multiple paternity in the rainy season than in the preceding dry season  horei, females do not form pair bonds with their mates, but may successively spawn with different males and be targeted by sneaking males Below is the link to the electronic supplementary material.


Eln+/− and aged mice. Histological examination showed thinner internal and external elastic laminae, accompanied by increased elastin fragmentation in the medial layer without any calcium deposits in the small intrarenal arteries of kidneys from young Eln+/− and aged mice. Pressure myography of interlobar arteries showed that vessels from young Eln+/− and aged mice had a slight decrease in distensibility during pressure loading but a substantial decline in vascular recoil efficiency upon pressure unloading. To examine whether structural changes in the renal microvasculature influenced renal hemodynamics, we clamped neurohumoral input and increased renal perfusion pressure by simultaneously occluding the superior mesenteric and celiac arteries. Increased renal perfusion pressure caused robust changes in blood pressure in all groups; however, changes in renal vascular resistance and renal blood flow (RBF) were blunted in young Eln+/− and aged mice, accompanied by decreased autoregulatory index, indicating greater impairment of renal autoregulation. Finally, increased pulse pressure in aged Eln+/− mice positively correlated with high renal blood flow. Together, our data show that the loss of elastin negatively affects the structural and functional integrity of the renal microvasculature, ultimately worsening age-related decline in kidney function.Age-related decline in functional elastin is associated with increased arterial stiffness, a known risk factor for developing cardiovascular disease. While the contribution of elastin insufficiency to the stiffening of conduit arteries is well described, little is known about the impact on the structure and function of the resistance vasculature, which contributes to total peripheral resistance and the regulation of organ perfusion. In this study, we determined how elastin insufficiency impinges on age-related changes in the structure and biomechanical properties of the renal microvasculature, altering renal hemodynamics and the response of the renal vascular bed to changes in renal perfusion pressure (RPP) in female mice. Using Doppler ultrasonography, we found that resistive index and pulsatility index were elevated in young  Age-related remodeling of the arterial vasculature often manifests as decreased compliance and increased stiffness and is most notable in conduit vessels, including the aorta and carotid arteries . VasculaEln+/− mice) to demonstrate that elastin insufficiency alters the structure and function of the renal microvasculature and that these changes precede the development of hypertension  and elastin heterozygous (Eln+/−) mice that have been backcrossed more than ten generations into the C57BL/6 genetic background (Charles River). The generation of Eln+/− mice has been described previously  of Case Western Reserve University, School of Medicine. Experiments were performed using young (4–6-month-old) and aged (14–16-month-old) female wild-type . Images of renal arteries were acquired in B-mode with color Doppler setting to identify arterial blood flow. Peak systolic velocity (PSV), least diastolic velocity (LDV), and mean velocity (MV) determined from the renal Doppler waveforms were used to calculate resistive index  experiments were performed as previously described . In brieKidneys harvested from terminal renal hemodynamics experiments were cut into two in cross section and fixed in 10% formalin solution. Tissue embedding was performed at the Tissue Procurement Core at University Hospitals, Cleveland, OH, and sectioning and histological staining were performed at Cleveland Clinic, Cleveland, OH. Kidneys were stained with Movat pentachrome dye for elastic fibers and Von Kossa dye for the presence of calcium deposits. Scoring for elastin fiber fragmentation was achieved by blinding participants who scored the vessels based on the fragmentation on a scale of 1-3, with 3 being the highest abundance of fragmentation. Vascular calcification was then evaluated histomorphometrically at ×100 magnification in bright field. Double-blind assessment of vascular calcification was performed on random sections of aorta by separate investigators. The degree of Von Kossa positivity was scored semi-quantitatively, with scores ranging from 0 to 3, depending on the surface of Von Kossa positivity. A score of 0 indicated no Von Kossa positivity; a score of 1, focal Von Kossa positivity, larger than or not overlying a cell nucleus; a score of 2, partially circumferential Von Kossa positivity in the tunica media of the vessel; and a of score of 3, Von Kossa positivity in the tunica media spanning the complete circumference of the vessel.i) and external (De) diameters were calculated using the following equation: Histological measurements were completed using NIH ImageJ software. Measurements were obtained by manually tracing the internal perimeter (lumen) and external perimeter of the vessels. The internal  as previously described -N,N,N′,N′-tetraacetic acid (EGTA) with a pH of 7.4. The renal medulla was removed to visualize the renal microvascular tree, including segmental, interlobar, and arcuate arteries. After careful dissection and excision, interlobar artery segments were mounted on glass pipettes in a pressure myography chamber  and secured with two pieces of silk ligature at each end of the vessel. Both the vessel lumen and the vessel chamber were filled with Ca2+-free PSS, while the bath was warmed to 37°C before the start of the experiment. Intraluminal pressure and vessel bath temperature were maintained by servo-controlled pressure pump and temperature control systems, respectively. Vessels that failed to hold consistent pressure were considered leaky and were discarded. To measure passive loading diameter, intraluminal pressure was increased from 40 to 160 mmHg in 20-mmHg increments and maintained at each pressure for 1 min. To measure passive unloading diameter, intraluminal pressure was decreased from 160 to 40 mmHg in 20-mmHg decrements and maintained at each pressure for 1 min.Kidneys harvested from young and aged escribed . The com0 represents the internal diameter before increasing pressure, D1 is the internal diameter after increasing pressure, and ΔP is the change in intraluminal pressure which is 20 mmHg, as previously described , which represents the percent change in arterial internal diameter for each mmHg change in intraluminal pressure, was calculated as follows: escribed .post hoc test where appropriate. All values are presented as mean ± standard error of the mean (s.e.m). A p-value <0.05 was considered statistically significant.Statistical analysis was performed using Prism 9 . Multiple comparisons were performed using analysis of variance (ANOVA) followed by Sîdak Eln+/− mice, and these parameters appeared to be accentuated in aged mice, regardless of genotype. Both RI and PI values were elevated in young and aged Eln+/− mice and trended higher in aged Eln+/+ mice  and decreases (unloading) in intraluminal pressure to examine the overall arterial compliance. As shown in Eln+/+ and Eln+/− mice during pressure loading; However, in aged mice, Eln+/− interlobar arteries appeared slightly less distended relative to arteries from Eln+/+ mice at high intraluminal pressure levels  cocktail containing 2% bovine serum albumin, 1 μg/mL norepinephrine, 0.5 ng/mL arginine vasopressin, 0.2 mg/mL hydrocortisone, and 0.2 μg/mL aldosterone, as previously described , while there was a slight negative correlation between pulse pressure and RBF in aged Eln+/+ mice , calculated using values from real-time RBF recordings, trended lower in young Eln+/− mice but was elevated in aged mice of the same genotype (flow differences between Eln+/+ and Eln+/− mice remained the same even after occlusion of the superior mesenteric and celiac arteries to elevate renal perfusion pressure (Eln+/− mice display some level of impaired renal autoregulation that is worsened with age, accompanied by augmented impedance to blood flow.Finally, we determined whether the blunting of renal hemodynamic response to increased perfusion pressure in /+ mice . Calcula+/− mice . Prior tgenotype . The patpressure . TogetheElastin insufficiency in aging and disease is associated with increased vascular stiffness which, by itself, is an independent predictor of cardiovascular and renal diseases . PreviouThe renal vascular bed receives approximately 20% of total cardiac output and, under normal physiological conditions, maintains a steady blood flow by adjusting renal vascular resistance in response to changes in renal perfusion pressure . The affEln+/− and aged Eln+/+ mice had wider lumen diameter and thinner vessel wall, corresponding to decreased wall-to-lumen ratio. However, there was no evidence of calcium deposits as expected of vascular stiffening due to calcification (Eln+/− and aged mice. Thus, elastin insufficiency appears to accelerate age-related changes in the structural properties of the renal vascular bed. A major functional consequence of the age-related structural changes in the small arteries and arterioles of the kidney has been suspected to be the effect on the autoregulatory capacity of the renal vascular bed (Like the cerebral and uteroplacental vasculature, the renal vasculature is considered a low-impedance vascular bed, with high flow but high resistivity at the afferent arterioles . To the fication . Insteadular bed . Howeverin vivo assessment of the impact of age-related changes in the biomechanical properties of the renal microvasculature on renal autoregulatory response to an acute increase in perfusion pressure. Saturation of the circulatory system with kidney-specific exogenous neurohumoral factors, by continuous infusion, ensures that cognate receptors become desensitized and unresponsive to additional surge upon changes in renal perfusion pressure or blood flow (ex vivo pressure-diameter tests using isolated renal interlobar arteries. Consistent with this postulation, a direct assessment of the correlation between vascular distention and recoil showed that elastin insufficiency and aging similarly worsened vascular hysteresis in renal interlobar arteries. Thus, the findings demonstrate that decreased compliance of the renal microvasculature due to the loss of functional elastin contributes to age-related decline in renal autoregulation that may predispose to barotraumatic injury. Furthermore, our results suggest that the need for compliance, conferred by elastic fibers, for vascular recoil during the cardiac cycle, is not limited to conduit arteries but is equally relevant in low-impedance, high-flow vascular beds, including the renal vascular bed.Here, we used an experimental paradigm that enabled ood flow . Thus, iEln+/− mice. The results demonstrate that elastin insufficiency plays a significant role in the functional and structural changes associated with aging in the resistance vasculature of the kidney. Thus, the structural and functional features of the renal vascular bed in young Eln+/− mice are indicative of accelerated renal vascular aging.In summary, we report that alterations in renal hemodynamics that manifest in aging, as seen in aged wild-type mice, are already present in young


Most computational methods that infer somatic copy number alterations (SCNAs) from bulk sequencing of DNA analyse tumour samples individually. However, the sequencing of multiple tumour samples from a patient’s disease is an increasingly common practice. We introduce Refphase, an algorithm that leverages this multi-sampling approach to infer haplotype-specific copy numbers through multi-sample phasing. We demonstrate Refphase’s ability to infer haplotype-specific SCNAs and characterise their intra-tumour heterogeneity, to uncover previously undetected allelic imbalance in low purity samples, and to identify parallel evolution in the context of whole genome doubling in a pan-cancer cohort of 336 samples from 99 tumours. In the course of cancer evolution, cancer genomes frequently change the number of copies of their chromosomes or parts thereof. These changes in copy number can be estimated from DNA sequencing data, but current tools can typically not identify which parental copy of a chromosome the copy number change occurred on. We here introduce Refphase, a new algorithm and software tool for assigning copy number changes to the parental chromosomal copy they have occurred on. This assignment allows us to measure differences between cancer genomes in much more detail and allows us to systematically catalogue different types of evolutionary events, including the detection of parallel evolution where highly similar genetic changes occur in different parts of the evolutionary tree simultaneously. As an example, we show how after whole-genome duplication successive losses of chromosomes affect different parental copies in different regions of a tumour, reducing the average copy number from four copies to close to three copies independently. As a consequence of genomic instability, cancers accumulate somatic mutations . These iSCNAs are typically identified in an allele-specific manner from DNA sequencing or single nucleotide polymorphisms (SNP) array data using two measures: the log read-depth ratio (LogR) of a genomic locus between the tumour and a matched normal sample, which informs total copy number estimates; and the B-allele frequency (BAF) at heterozygous SNPs, which informs allelic imbalance estimates . BAF andAdditionally, while BAF and LogR allow inference of allele-specific SCNAs, the resulting copy number profiles and underlying SNPs are often not phased, meaning that SCNAs cannot be assigned to the physical haplotypes they reside on. Instead, allele-specific copy number states are typically reported in an unphased major/minor configuration, where major and minor refer to the greater and lesser copy number at a genomic locus respectively. However, phasing of SCNAs can offer additional insights into the clonality of mutational events, giving resolution on whether the same parental allele has been gained or lost across different samples in a single tumour.Statistical phasing utilises large collections of genotypes  and locamulti-sample reference phasing .As the assignment to haplotype A and B is arbitrary across chromosome boundaries, for plotting purposes, we flip the A and B allele assignment for all samples on a chromosome-by-chromosome level to ensure that on average haplotype A has a higher copy number than haplotype B.Following reference phasing, Refphase uses the re-estimated sample ploidies, input purities and segment-level LogR data to call SCNA events in each sample .We consider an SCNA to be a deviation of any length from the diploid major/minor copy number state of . SCNAs with an unequal number of copies on both parental alleles  are termed allelic imbalance.amplifications, gains and losses relative to ploidy as well as LOH events and homozygous deletions. Events are called for each segment and sample independently of others by comparing the mean segment LogR distributions to calculated purity-ploidy derived event thresholds  is called for segments in which the major rounded integer copy number state equals the rounded tumour sample ploidy.Finally, a tumour-level event summary is calculated .Inter-sample heterogeneity is quantified. The presence or absence of each class of relative-to-ploidy event, loss of heterozygosity (LOH), and homozygous deletions (HDs) in each tumour sample for each minimum consistent segment is noted. This presence or absence classification is then examined in the context of MSAI detection to determine whether each event affects the same allele in all samples.For a given segment, an SCNA is considered to be clonal if it is present in every sample and affects the same allele in all samples. An SCNA is assigned as subclonal if it is present in at least one sample but simultaneously absent in at least one other sample . Crucially, in cases in which the same relative-to-ploidy or LOH event type is determined to occur in every sample of a given tumour but in the context of MSAI such that a different allele is deemed to be the major allele in different samples at the same segment, the event is assigned as subclonal and parallel and not as clonal, since the SCNAs in different samples are deemed to be of different origin and affecting different haplotypes. Specifically, a mirroring of alleles must be observed between the specific samples harbouring the event type of interest for the event to be called parallel. For example, in a tumour composed of three samples, should a gain SCNA be called in Samples 1 and 2 only, the major alleles must differ in Samples 1 and 2 for the gain event—already subclonal—to be called parallel, regardless of allelic arrangement in Sample 3.Having assigned individual SCNAs as clonal or subclonal, the proportion of the genome affected by each specific event type  is calculated. Proportion of genome measures are calculated over the sum of minimum consistent segment widths for a tumour.Plotting options are available to visualise tumour-level summary metrics and the degree of reassignment of events from clonal to subclonal based on MSAI context.Refphase also offers the option for a user to define subgroups of samples and calculate group-level (instead of whole-tumour-level) summary metrics and create within- and between-group summary plots. Examples of primary-sample-specific and metastases-sample-specific summary tracks in which clonality and heterogeneity analyses are restricted to the respective subgroups of samples are shown in Figs The phylogeny showcased in et al. [et al. [In order to derive the WGD status for the Brastianos et al.  and Sott [et al.  datasets [et al. . SubsequASCAT  allelic CNLOH events were simulated in chromosomal segments from NSCLC multi-sample bulk sequencing from the TRACERx100 cohort .A the copy number state of allele A and nB the copy number state of allele B.First, Refphase was run on the TRACERx100 tumour samples. Then, pairs of samples were selected from tumours where at the same genomic region, defined by a Refphase bin, one sample, referred to as “reference”, demonstrated allelic imbalance allowing a reference phasing to be obtained and the other sample, referred to as “test” demonstrated total copy number equal to the overall sample ploidy with no allelic imbalance. Specifically, for a genomic bin, the reference sample demonstrated sufficiently clear allelic imbalance in its BAF to be likely to produce a highly accurate phasing if the heuristic condition in Additionally, for candidate samples and segments to be chosen, the following conditions must also be satisfied: Genomic bins had to be ≥1/5 of the size of the chromosome on which they were located and contain ≥1/5 of the heterozygous SNPs present on the same chromosome; the ploidy of the test sample had to be between 1.8 and 2.2 or between 3.8 and 4.2 (lower and upper bounds inclusive); and the total rounded copy number in the genomic bin should equal the total rounded ploidy of the test sample.This candidate segment selection approach produced 125 segments that were then used to simulate CNLOH at the genomic region in the test sample. CNLOH was simulated at various cancer cell fractions (CCFs) of the sequenced sample, ranging from 1% to 30% in steps of 1%. Specifically, BAF values were assigned at each of the heterozygous SNP positions in candidate segments using a binomial distribution (R rbinom function) with simulated probability equal to the mean BAF which would be predicted for each phased allele at the segment based on copy number and simulated purity and with the number of trials equalling a simulated sequencing coverage of 200(x).These simulated BAF profiles are then used as input to ASCAT allele specific piecewise constant fitting segmentation and Refphase, making use of the phasing derived reference sample, and detection of allelic imbalance reported for each method independently for each of the 125 candidate segments at each simulated CCF value. Detection of allelic imbalance was reported as detection of a CNLOH event.Tumour cellularity and ploidy for each sample assayed with SNP arrays were estimated using the ASCAT algorithm . ASCAT wAll datasets downloaded were processed from FASTQ files using a previously described pipeline ,9. TumouTo test whether MSAIs were more frequently found than expected by chance, we created a simulation approach . To thisTo enable comparison with Battenberg and HATCHet, we removed Refphase’s haplotype-specificity by simply designating the major allele copy number and minor allele copy number for each segment in Refphase output as the higher and lower copy number respectively, on a per sample basis. This masks mirrored subclonal allelic imbalance detection but leaves total copy number estimates unchanged.For the purposes of benchmarking we used only a single full genome-wide copy-number profile from each algorithm for each sample. For Refphase, this was its standard output modified as described above to remove mirrored subclonal allelic imbalance detection. However, for HATCHet and Battenberg, the genome-wide copy-number profile represented only the dominant subclone per sample comprising both clonal SCNAs and those subclonal SCNAs present in the highest fraction of tumour cells within that sample. We then compared the intersection of copy number segments from all three algorithms.To generate artificial normal samples, we used the BAF values from the matched normal sample of the Brastianos tumour under investigation. The Brastianos samples were sequenced to median average depth of 108.3(x). These BAF values were previously not being used for any calculation, since the matched normal sample is only used as a read depth reference to compute logR values. To generate a new logR track for these samples, we utilised two distinct sequenced normal tissue samples from the same patient from the TRACERx-421 cohort . These tS1 FigThe reference phasing algorithm phases individual segments across all samples but not segments with respect to each other. Therefore we employ a horizontal phasing algorithm which minimises the total number of copy-number breakpoints defined as the total Hamming distance between neighbouring segments . To this(PDF)Click here for additional data file.S2 FigThe upper three panels show tracks for log read-depth ratio (LogR), B-Allele Frequency (BAF), re-estimated fractional copy number states, and somatic copy number aberration (SCNA) event calling at a sample level. The bottom panel (‘Summary’) gives a tumour-level summary of SCNA event clonality and detection of mirrored subclonal imbalance (MSAI), loss of heterozygosity (LOH) and parallel events. Events are called relative-to-ploidy using a mean logR threshold.(PDF)Click here for additional data file.S3 Fig(a) allelic imbalance (LME coefficient = 0.10 (2.s.f.), LME ANOVA p = 0.03), (b) newly identified allelic imbalance (LME coefficient = -0.28 (2.s.f.), LME ANOVA p<0.0001), (c) allelic imbalance, as defined by Refphase and using original estimates from ASCAT , (d) MSAI (LME coefficient = 0.0045 (2.s.f.), LME ANOVA p = 0.5). (e) The association between mean tumour purity and proportion of genome with MSAI (LME coefficient = 0.070 (2.s.f.), LME ANOVA p = 0.07). Proportion of genome data is calculated using Refphase. Analyses are undertaken for the 336 tumour samples from 99 tumours in the pan-cancer cohort described in The association between sample purity and proportion of genome with (PDF)Click here for additional data file.S4 Figa) An example of the effect of decreasing tumour purity on BAF band separation. b) The effect of varying copy number states on BAF band separation for a tumour of fixed purity, here 50%. c) Example of typical BAF and LogR profiles for a loss of heterozygosity (LOH) with total copy number loss (left) and copy-neutral LOH (CNLOH) (right). Relative-to-ploidy thresholds are indicated by dashed lines and are derived for a diploid 100% pure tumour using the formulae described in the Methods of this manuscript. Orange and blue points throughout plots represent the phased “A” haplotype and “B” haplotype respectively.(PDF)Click here for additional data file.S5 Figa) Proportion of the genome subject to subclonal SCNAs . b) Proportion of aberrant genome subject to subclonal SCNAs, where aberrant genome is defined as the total length of genomic segments in which any SCNA event  is called . c) Proportion of genome subject to clonal loss of heterozygosity (LOH) . d) Proportion of the genome subject to subclonal LOH . All p-values shown are for paired Wilcoxon signed rank tests with continuity correction, across the n = 99 tumours described in Figs (PDF)Click here for additional data file.S6 FigMedian proportions by Patient WGD Status . Kruskal-Wallis p-value is shown (p = 2e-04). Data is shown for n = 99 tumours from the pan-cancer cohort showcased in (PDF)Click here for additional data file.S7 Figa) Examples of parallel gain events on chromosome arm 7p, observed between glands on different sides of the tumour. b) Example of a parallel gain event on chromosome 2, observed within glands on the same side of the tumour. Tumour IDs shown match those in the original publication [lication .(PDF)Click here for additional data file.S8 FigWe ran MEDICC2 to infer the mutational events that occurred for each sample of the Brastianos and Sottoriva datasets, and used these events as input to a simple karyotype simulator. The simulation uniformly chooses a haplotype to apply the event to, and uniformly samples the starting locus for segmental events. The available phylogeny is then simulated, and the proportion of MSAI segments calculated . Performing 350 simulations per sample, we generate the above violin plots in the same style as (PDF)Click here for additional data file.S9 Figa) number of samples , and b) tumour purity difference—defined as the purity difference between the most and least pure sample within a tumour . Proportion of aberrant genome is defined as the proportion of the total length of genomic segments harbouring any relative-to-ploidy SCNA (gain or loss) or loss of heterozygosity (LOH) event which contains a subclonal SCNA or LOH event. Proportion of the aberrant genome is defined at the tumour level. Analyses are carried out for the pan-cancer cohort described in Figs et al. study [et al. study [Association between proportion of aberrant genome with l. study ; SNP arrl. study . Best fi(PDF)Click here for additional data file.S10 Figa) total copy number across all segments b) total copy number across all segments without within-sample subclonal copy number called by both Battenberg and HATCHet c) total copy number across all segments that demonstrate within-sample subclonal copy number called by both Battenberg and HATCHet.d) Major copy number across all segments e) Major copy number across all segments without within-sample subclonal copy number called by both Battenberg and HATCHet f) Major copy number across all segments that demonstrate within-sample subclonal copy number called by both Battenberg and HATCHet. g) Minor copy number across all segments h) Minor copy number across all segments without within-sample subclonal copy number called by both Battenberg and HATCHet i) Minor copy number across all segments that demonstrate within-sample subclonal copy number called by both Battenberg and HATCHet.Barcharts for each sample in the Gundem et al.  WGS coho(PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.


This narrative review provides an overview of the current advances, challenges, and opportunities in nanoparticle drug delivery for central nervous system (CNS) disorders. The treatment of central nervous system disorders is challenging due to the blood-brain barrier (BBB), which limits the delivery of therapeutic agents to the brain. Promising approaches to address these issues and improve the efficacy of CNS disease therapies are provided by nanoparticle-based drug delivery systems. Nanoparticles, such as liposomes, polymeric nanoparticles, dendrimers, and solid lipid nanoparticles, can be modified to enhance targeting, stability, and drug-release patterns. They allow for the encapsulation of a variety of therapeutic compounds and can be functionalized with ligands or antibodies for active targeting, minimizing off-target effects. Additionally, nanoparticles can circumvent drug resistance processes and provide versatile platforms for applications that combine therapeutic and diagnostic functions. Although the delivery of CNS medications using nanoparticles has advanced significantly, there are still challenges to be resolved. These include understanding the BBB interactions, doing long-term safety studies, and scaling up the production. However, improvements in nanotechnology and a deeper comprehension of CNS disorders provide opportunities to enhance treatment results and address unmet medical requirements. Future research and ongoing clinical trials are required to further explore the potential of nanoparticle drug delivery for CNS disorders. The treatment of central nervous system (CNS) disorders poses significant challenges due to the blood-brain barrier (BBB), which restricts the passage of most therapeutic agents from systemic circulation to the brain. Over the years, nanoparticle (NP) -based drug delivery systems have emerged as promising solutions to overcome these challenges and improve the efficacy of CNS disorder treatments. Nanoparticles offer unique advantages, such as enhanced drug stability, prolonged circulation time, and the ability to encapsulate a wide range of therapeutic agents. This narrative review provides an overview of the current advances, challenges, and opportunities in nanoparticle drug delivery for CNS disorders.The BBB functions as a selective barrier, preventing noxious substances from entering the brain while controlling the passage of necessary nutrients and drugs. The delivery of therapeutic medications is nevertheless restricted by its tight junctions and efflux transporters. Drugs can be delivered to the brain effectively by using nanoparticles that can be created to avoid or take advantage of these obstacles . For CNSNanoparticles offer the ability to encapsulate a wide range of therapeutic agents, including small molecules, proteins, nucleic acids, and even gene-editing tools. This adaptability makes it possible to give various medications in a single formulation, use combination therapy, and employ personalized medicine techniques . AdditioNanoparticles can also circumvent multidrug resistance mechanisms in CNS disorders by modulating drug release kinetics, co-delivering efflux pump inhibitors, or utilizing alternative drug delivery routes to bypass resistant cells . They haNanoparticle-based drug delivery systems have shown promise for various CNS disorders. Future therapeutic uses for these technologies are possible, as shown by ongoing clinical trials testing their effectiveness and safety. Although nanoparticle-based CNS medication delivery has made great progress, there are still several obstacles to overcome. For nanoparticle formulations to be successfully translated into clinical practice, further research is required to understand how the BBB interacts with them, conduct long-term safety analyses, and scale up manufacturing. However, the ongoing advancements in nanotechnology and the growing understanding of CNS disorders offer tremendous opportunities for improving therapeutic outcomes and addressing unmet medical needs. Opportunities in NP drug delivery for CNS disordersEnhanced Permeation and Retention EffectNP-based drugs are helpful, especially because of their ability to permeate the BBB to a greater extent as compared to traditional strategies. Their ability to cross the BBB depends upon their size, shape, surface chemistry, and stability. NPs getting trapped in selected sites like tumors constitute a passively targeted drug delivery system. This phenomenon is named the enhanced permeation and retention effect (EPR effect) . The reaActive Targeting StrategiesIf we want to achieve maximum efficacy from NPs with limited side effects, we must use techniques to specifically target the cells of the brain requiring treatment. Both non-invasive and invasive techniques have been postulated. Non-invasive biologic methods like antibody-directed, receptor-directed delivery and chemical methods like using a drug conjugate are possible solutions to employ targeted approaches. Invasive ways of targeting can include intracerebral implants . CurrentResponsive Drug ReleaseOne way to achieve better efficacy in drug delivery with minimal side effects is to practice stimulus-responsive drug release. It means the NPs loaded with the drug will deliver it to the site that provides it with a specific stimulus. The trigger for drug release can be physical, chemical, or biological. Various stimuli like a particular pH at a site, temperature, enzyme activity, redox potential, and light, etc. have been explored. It is well known that tumors create a microenvironment different from the physiologic state. They may exhibit different pH, oxygenation, perfusion, and metabolic state. We can utilize the change of pH in the tumor microenvironment by formulating NPs using a pH-sensitive phospholipid component. Phosphatidylethanolamine (PE) or oleic acid (OA) are widely used substances for conferring pH sensitivity to liposome NPs . AlternaMultimodal Imaging and Therapeutic AgentsNPs with combined imaging and therapeutic properties are of special interest to scientists these days. Particularly, magnetic NPs (MNPs) are being extensively explored to this end. A study has demonstrated positive results in using MNPs with an iron-cobalt core and a graphite carbon shell (FeCo/C), delivering small interfering RNA (siRNA) to tumor cells . In addiPersonalized Medicine ApproachesNanotechnology is paving the way for making personalized medicine a possibility soon. The idea is to use NPs to detect the tumor biomarkers, responsible for the pathogenesis or clinical outcomes in a specific patient, and simultaneously load them with some drug (i.e. using siRNA) which will alter the expression of those tumor biomarkers .NP-based drug delivery systems for CNS disordersNanoparticle-mediated drug delivery systems for CNS disorders encompass a range of formulations, such as lipid-based, polymeric, and metallic nanoparticles Figure .Lipid-Based NPs Liposomes, solid lipid NPs, nanostructured lipid carriers, and nano-emulsions are some of the types of lipid-based NPs -16. TheyLiposomes can vary in size and form unilamellar or multilamellar structures. Their membranes are made of two layers of phospholipids and they can be loaded with drugs . Their sSolid Lipid NPs (SLNs) are solid core lipid nanostructures capable of carrying both hydrophilic and lipophilic drugs . They coSLNs have some drawbacks like drug expulsion during storage, insufficient carrier loading, and large water content. To overcome these, nanostructured lipid carriers (NLCs) were introduced which are a modified version of SLNs. While SLNs have only a solid core, NLCs comprise both solid and liquid lipids .Polymeric NPsPolymer-based NPs offer some beneficial characteristics that make them a good candidate for an efficient drug delivery system. They have low toxicity, are biocompatible, and enable regulated drug release . Among pChitosan (CS), an alkaline polysaccharide derived from chitin, has been used in nanotechnology due to its biocompatibility and easy availability. The presence of active hydroxyl and amino groups in chitosan allows us to introduce different functional groups and modify them according to our requirements. It is believed that chitosan also has some bacteriostatic, fungistatic, spermicidal, and anti-carcinogenic properties. CS NPs have some limitations like their low solubility at neutral and alkaline pH i.e. physiologic pH. This causes low bioavailability due to pre-systemic metabolism after oral intake . Dendrimers are three-dimensional polymeric nanostructures with radially symmetric molecules having tree-like branches. The most commonly used dendrimers are the ones derived from polyamidoamine. Their surface can be modified to have various functional groups like hydroxyl, amino, or acid groups. These groups can be conjugated with targeting ligands like Folic acid, allowing us to produce intelligent NPs against specific tumor sites . DendrimMetallic NPsGold NPs (GNPs or AuNPs) can carry small drugs or large molecules like DNA or proteins. Their size can be tuned according to the payload features. These NPs have the advantage that the gold core is nontoxic. GNPs have an added benefit due to their interaction with thiol groups, which allows for controlled intracellular release . DendrimMagneto-electric NPs (MENPs) are the latest advancement in nanomedicine with promising future applications. MENPs can work as a two-way interface at the molecular level . They caApplication of NP drug delivery for CNS disordersAlzheimer’s DiseaseConventional orally administered drugs used in neurodegenerative diseases have to pass through first-pass metabolism, crossing BBB and sustainable circulation in the bloodstream. Characteristics of drugs like polarity, molecular weight, net charge, solubility, and affinity for hydro or lipid moieties are mainly responsible for therapeutic failure. The NP drug delivery system modulates these properties and enhances the therapeutic potential of delivered drugs through this system. Amyloid [Aβ] deposition is a major pathophysiologic factor in the severity of Alzheimer’s disease. A study confirmed that when functionalized liposomes conjugated with phosphatidic acid and cardiolipin they could effectively target these amyloids with the highest affinity . AnotherParkinson’s DiseaseNanotechnology offers a promising future for the treatment of Parkinson’s disease. Dopamine-loaded liposomes were directly implanted in the striatum of a rat and it was found that the levels of dopamine remained elevated for almost 25 days with 40 days of continuous dopamine release after the implantation . Other sMultiple SclerosisThe use of nanotechnology for the treatment of multiple sclerosis is based on its ability to target immune cells and create immune tolerance. NPs can also encapsulate some disease-specific auto antigens and present them to antigen-presenting cells in a way that the resultant immune response is tolerable by the patient -40. ThisBrain TumorsSmall interfering RNA and micro RNA are two examples of naked nucleic acids that have the potential to treat gliomas very effectively but their use is limited by their fast degradation, poor internalization, and lack of specificity. NPs were found to prevent this fast degradation and promise a future use of these naked RNA molecules in the treatment of gliomas . NPs areTraumatic Brain Injury and Stroke The altered pH in a stroke can be used by stromal cell-derived factor-1 alpha-loaded, pH-sensitive polymeric micelles to create a favorable microenvironment for neuronal recovery . TargetePreclinical studies A preclinical tryout was conducted in India on nano lipid carriers containing curcumin delivery into CNS by the intranasal route. The objectives of the study were to enumerate its cytotoxic effects and assess its efficacy. The NP curcumin-loaded nano lipid carriers were produced in this study by hot high-pressure homogenization technique. A surfactant and stabilizer molecule were also used which were Tween 80 and lecithin, respectively. Curcumin was released in a biphasic pattern that was a burst release initially followed by a constant rate . It was Another preclinical trial synthesized 131I-labelled multifunctional dendrimers/NPs for targeted tumor radiotherapy and single-photon emission computerized tomography (SPECT) imaging. These NPs were formed by conjugation of carboxylic acid functionalized methoxyl polyethylene glycol (mPEG-COOH), chlorotoxin conjugated polyethylene glycol (PEG-CTX), and 3-(4'-hydroxyphenyl) propionic acid-Osu (HPAO) onto amine-terminated Generation 5 polyamidoamine (G5 PAMAM) dendrimers. It was found that CTX-targeted multifunctional dendrimers had increased specificity for gliomas in comparison to other dendrimers. This specificity also allowed efficient SPECT imaging. The CTX-PEG was compared with the other two dendrimers, methoxypolyethylene glycol maleimide (PEG-MAL), and Na-131 to find out the radioactive inhibition of the tumor. The CTX-PEG dendrimer exhibited the highest tumor suppression in comparison to the other two .Gold NPs were used in a preclinical trial to find out their neuro-protective effect on Parkinson’s disease. In this study PC12 cells were synthesized that are rat-derived cell lines and can differentiate into neuron-like cells. It was found that these gold NPs can prevent apoptosis of these PC12 cells thus halting the progression of Parkinson’s disease. The Nissl bodies of the mice on which the experiment was conducted, illustrated a therapeutic effect on nerve cell damage. There were no significant toxic effects found of these gold NPs on tissues on histological examinations of the heart, spleen, lung, kidneys, and brain. It was found that the highest concentration of these gold NPs was found in the spleen which is an indication of their effective clearance . Pituitary acetylates cyclase-activating peptide (PACAP); a peptide that has a neuro-protective function was activated in mice by using gH26 liposomal NPs in a preclinical tryout. The study concluded that regular liposomes and liposomes conjugated with gH26 had no significant difference in the integrity of BBB however initially within 30 minutes and at higher concentrations of PACAP activating peptide gH26 liposomes were found to be more efficient in the transport of PACAP. Furthermore, it was found that gH26 liposomes had no effect on the integrity of BBB and hence had no toxic effects on the brain . Clinical studies A phase 2 clinical trial for glioblastoma using pegylated liposomal doxorubicin for prolonged delivery of temozolomide and radiotherapy showed a 30.2% survival rate with a 12-month progression-free period. The toxicity of temozolomide with this NP was also found to be tolerable in patients. However, neither the administration of this NP nor the prolonged administration of temozolomide had a significant impact on the patient’s outcome . Side effects, tolerability, and safety of gadolinium-based NPs, AGuIX in combination with standard complete brain radiotherapy was observed in a phase 1 clinical trial in patients with multiple brain metastases. The study concluded that these NPs are capable of accumulating in brain metastases thus increasing the effectiveness of radiotherapy . This stFuture directions and recommendationsThe future developments and potential risks of NP drug delivery for CNS pathologies are becoming increasingly important. Different factors such as size shape and surface properties play a critical role in achieving the best optimization for NPs. Altering of surface moieties (e.g. polyethylene glycol) and size can reduce the premature clearance of NPs which can lead to the accumulation of these agents in tumor cells/sites . NPs smaMany targets are identified by NPs in CNS. Recent literature suggests that low density lipoprotein receptor-related protein 1 (LRP-1) is the target of NPs which is upregulated in brain tumors and is present in the capillary endothelium of BBB . Target Safety assessment of NPs is quite essential to consider before increasing its clinical use. Many factors such as the specific surface area of these particles can cause adverse effects such as the formation of reactive oxygen species . HoweverA clinical trial conducted on mice demonstrated that functionalized NPs that were combined with 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG2K) could cross BBB easily in comparison to non-functionalized NPs or unconjugated NPs . For the management of ischemic stroke, a clinical trial was performed by administering sequential therapies of NPs with anti-inflammatory and angiogenesis properties respectively by using a hydrogel to deliver vascular endothelial growth factor (VEGEF) and the ant-inflammatory drug 6-bromoindirubin-3′-oxime (BIO) for angiogenesis. The trial found that this sequential release of anti-inflammatory NPs followed by BIO NPs was more effective than administering both NPs separately . The devFor regular use of NP drug delivery, a proper regulatory system for nanotechnology must be considered. Clinical and preclinical tryouts must be performed continuously to identify adverse effects and improve their efficacy. Quality control agencies must be employed for monitoring the production of nano-based drugs and robust quality control measures must be ensured. Along with these post-market surveillance team must also be recruited to ensure proper market handling of these products. Liposomes, polymeric nanoparticles, dendrimers, and solid lipid nanoparticles are a few examples of the several types of nanoparticles that have been created and may be altered to improve targeting to certain brain regions, increase stability, and optimize drug release patterns. Opportunities in nanoparticle drug delivery for CNS disorders include leveraging the enhanced permeation and retention effect, employing active targeting strategies, achieving responsive drug release, developing multimodal imaging and therapeutic agents, and utilizing personalized medicine approaches. Several obstacles still exist in nanoparticle-based CNS medication delivery, despite recent advancements. It is necessary to undertake long-term safety evaluations, scale up production for clinical translation, and better understand how nanoparticles interact with the BBB. A rising understanding of CNS illnesses and continuous advances in nanotechnology, however, provide tremendous possibilities to enhance therapy results and address unmet healthcare needs.


A 73-year-old woman with Parkinson’s disease (PD) underwent implantation of bilateral subthalamic nucleus deep brain stimulators (STN-DBS) to address bilateral upper extremity medication-refractory tremor. Post-operatively, she experienced a “see-saw effect” where small increases in stimulation resulted in improvement in one symptom (tremor) with concurrent worsening in another (dyskinesia).SID is usually considered a positive predictor of DBS outcome. However, there are cases where SID cannot be optimized. Lead location and pre-operative characteristics may contribute to this adverse effect. If the combination of programming and medication adjustments fails to resolve SID, what can be done to “rescue” the outcome?Management of SID requires a gradual and steadfast programming approach. Post-operative lead localization can guide advanced programming and decision-making. Rescue surgical interventions may be considered.In cases where SID is persistent despite deploying persistent optimization strategies, there is limited guidance on next steps. A 73-year-old woman with a 23-year history of tremor-predominant PD presented for evaluation of refractory tremors which co-occurred with medication-induced dyskinesia despite exhaustive attempts at adjusting medication types, timing and dose. She underwent bilateral subthalamic nucleus (STN) deep brain stimulation (DBS) at the University of Florida Fixel Institute for Neurological Diseases Campus. The STN target was selected on the basis of interdisciplinary review and in conjunction with the patient’s priority of maximal and immediate tremor control. Her initial post-operative monopolar programming threshold review  revealedConsistently, she reported SID at electrical current levels that were applied below 1 mA. The location of her DBS lead and active contacts were calculated using co-registration of pre-operative volumetric MRI and a post-operative high resolution CT scan ) . Lead loThis case demonstrates the potential for STN-DBS to result in persistent SID. Initial post-operative counseling of this patient focused on the usual favorable outcome associated with SID. However, a “see-saw effect” can emerge where small increases in stimulation result in improvement in one symptom (tremor) with concurrent worsening in another (dyskinesia). Alternatively, reduction in medication led to worsening of tremor and improvement in dyskinesia. The clinician may thus apply programming and medication strategies but persistently be unable to balance the clinical see-saw. The question in this case involves the potential impact of underlying disease characteristics, the impact of lead location and the region of stimulation, and weighing the value of continued stimulation that produces SID. This guides shared decision making around consideration of a rescue GPi DBS, lead relocation, or a pallidotomy procedure.Managing SID is usually approached with the clinician employing slow ramping of stimulation amplitude over several months. Many experts minimize pulse width and frequency, focusing on deploying stimulation to more dorsal regions on the DBS lead. Additionally, directional programming, bipolar stimulation, tripolar stimulation, and interleaving may be trialed. The imaging in this case revealed a relatively ventral and anterior location for both DBS leads. The team used the measured lead localization to “steer current” into a potentially more favorable and more dorsal lateral location for potential optimal neuromodulation. In this case, the programming was complex, comprehensive and time consuming.Ultimately, a combination of double monopolar and directional stimulation with a reduced pulse width and a reduced frequency was employed . FrustraThe post-operative UPDRS part III motor score at the initial programming session, one month following lead placement was 48 off medication and off stimulation. Her 6-month post-bilateral optimized DBS UPDRS part III motor sub-score was 45 off medication/off stimulation and 13 on medication/on stimulation.A video showing her response to DBS and medications is provided  as is a Due to her persistent, bothersome SID, she was offered and is considering a staged approach to adding “rescue” GPi leads, beginning with the left side for the more bothersome right hemibody. This could be done either by leaving the current leads in place or removing and replacing them with leads in the GPi target.Dyskinesia associated with STN-DBS has been widely reported in PD. The dorsolateral and posterolateral regions of applied stimulation have been postulated to underpin the effect, however there are few cases with details for subjects with persistent SID 2. NotablDyskinesia may occur in other non-PD related hyperkinetic movement disorders. In one recent series of patients with Meige syndrome who were treated with STN-DBS, SID was associated with more ventral (including substantia nigra pars reticulata) rather than the dorsal regions usually observed in SID PD cases. Though the study methods were suboptimal in the application of STN segmentation and relied on stereotactic coordinates, they do raise the important question of influence of the underlying disorder on the SID . Support57Perhaps the best chance to avoid SID may be the recognition of potential pre-operative risk factors, especially “brittle dyskinesia” (hyperkinesia resulting from the use of ≤ one 25/100 mg tablet of carbidopa/levodopa per dose). Once persistent SID emerges, using empirical trials of dorsal stimulation, bipolar, tripolar or directional stimulation all may provide potential management solutions 10. RampiMany experts may be swayed to use STN DBS if tremor suppression is the primary goal. However, a recent meta-analysis of STN and GPi DBS has revealed similar tremor suppression outcomes and thus it may be more important to consider “brittleness” and how disabling dyskinesia is pre-operatively 13. There312This case underscores the importance of recognizing persistent SID and the “see-saw dilemma” which may emerge during management. There is a paucity of published studies to guide DBS programming and to advise concurrent medication adjustments. The see-saw effect  refers tGPi is a well-known dyskinesia suppressor. Well-placed leads in the GPi target afford the managing clinician greater latitude in the administration of higher doses of levodopa, while simultaneously “ramping up” the total amount of stimulation delivered. The clinicians in this case did their best to manage persistent STN SID through slow ramping of stimulation over time, dorsal stimulation, bipolar stimulation, tripolar stimulation and attempts at applying a directional DBS strategy. In our experience, some cases of SID will resolve with the application of slow ramping (e.g. 0.1–0.2 mA per week).Even when employing these strategies, there are instances where STN lead revision or the addition of a GPi rescue lead will be necessary. In the case of suboptimal lead location, the risks and benefits of repositioning the STN lead into a more dorsolateral position should be weighed. In some circumstances, STN stimulation may be producing partial benefit below the threshold for SID. In this circumstance, maintaining the contribution that STN stimulation provides, while adding a GPi lead to produce concurrent stimulation may aid in a patient’s overall therapy and offer more long term programming options. In most of these circumstances, when there is concern for losing positive STN benefit, maintaining the STN lead and adding a GPi lead, rather than revising the STN lead alone, is the safer and higher-yield approach to address persistent SID.In this case, the anterior and ventral location of the STN DBS leads could argue that repositioning dorsolateral may resolve the issue. Data supporting direct dyskinesia suppression with GPi stimulation may reduce clinical equipoise for a return to the STN target. Finally, this case adds a measure of circumstantial support to the argument that a baseline propensity for brittle dyskinesia should be considered when choosing a brain target (STN vs. GPi) during an interdisciplinary pre-operative DBS workup.


Early in the COVID-19 pandemic, nearly one in five adults in Canada worried about having enough food to meet their household’s needs. Relatedly, throughout the pandemic, public messaging repeatedly urged Canadians to support food charities, including food banks. Yet few studies have examined food bank usage during the pandemic or whether food charities were widely used by Canadians worried about food access.This study draws on four rounds of nationally representative surveying conducted during the COVID-19 pandemic between May 2020 and December 2021 among adults 18 years and older living in Canada. Descriptive statistics were used to examine rates of food-related worry during all four survey rounds. Data from the fourth survey round, collected in December 2021, were used to explore use of food-based community programs since the onset of the pandemic, including food banks. Logistic regression analyses were used to examine differences in socio-demographic and health-related characteristics between adults who did and did not report accessing food banks before and after adjusting for household income.n = 12,091), more than one in seven participants reported stress or worry related to having enough food to meet their household’s basic needs in the previous two weeks. Yet, by December 2021, fewer than 4% of participants reported ever accessing a food bank during the pandemic. Younger age, living with a child, financial concerns due to the pandemic, two different measures of food worry, pre-existing mental health conditions, disability, LGBT2Q + identity, and racialized or Indigenous identity, were each statistically significantly associated with higher odds of using food banks even when controlling for household income.Across survey rounds (Despite persistently high rates of food-related worry in 2020 and 2021 in Canada, relatively few adults reported accessing food banks or other charity-based community food programs. While respondents facing social, financial, and health-related inequities and reporting food worry were more likely to use food banks, most respondents did not report food bank use, regardless of financial or demographic circumstances or experiences of food worry. Findings align with previous research indicating that more adequate and comprehensive supports are needed to alleviate food-related-worry in Canada.The online version contains supplementary material available at 10.1186/s12889-023-16602-x. Since the onset of the COVID-19 pandemic in early 2020, there has been renewed interest from researchers, health professionals, policy makers, and the public in understanding the experiences of individuals who live in food insecure households . In CanaWhile the consequences of the most severe forms of food insecurity are clear, researchers have now demonstrated that milder experiences of food insecurity are also associated with poorer health. Survey respondents who answer affirmatively to even one question on the 18-item HFSSM survey module , are more likely to experience poorer self-reported mental and physical health than those with zero affirmatives on food insecurity survey scales –21. EmerSince 2020, income shocks following workforce layoffs, lost work hours, workers’ illnesses, and quarantine orders, all had the potential to affect income-related food worries; as did ensuing economic and supply chain consequences and the rapidly increased rates of food inflation that began in late 2021 in Canada , 28–30. Although national food insecurity monitoring in Canada using the HFSSM includes an item that relates to worrying about running out of food before having money to purchase more, few national studies during, before or since the onset of COVID-19 specifically report the prevalence or trends related to this specific experience of food insecurity. Still, data from the Canadian Community Health Surveys , CanadiaAs we build our understanding of how patterns of food-related worry and insecurity changed over the course of the pandemic, there is also a need for more nuanced understanding of the policy and programmatic responses that intersected with food-related experiences. Public discourse and media coverage during the pandemic frequently highlighted governmental and private investments in charity-based food programs e.g., –39). In . In 39])Despite the unprecedented infusion of public dollars, private donations, and media coverage in Canada highlighting the efforts of charity-based food programs to meet household food needs during the pandemic, there is little public oversight of charity-based food programs and no nationally representative public-use data to characterize the patterns of population-level usage of community-based food programs including food bank use. Estimates from before the pandemic suggest that less than a quarter of food insecure households in Canada reported drawing on food banks when income was limited , 55.worry in Canada during the pandemic, existing analyses have focused on the associations between such worries with mental health outcomes during the early stages of the pandemic  and Indigenous participants  at higher rates than non-racialized participants. Furthermore, participants who reported a pre-existing mental health concern , having a disability , or testing positive for COVID-19 at any time during the pandemic  had higher rates of food bank use compared to those not reporting these identities and experiences. The relationships between the socio-demographic and health-related variables remained statistically significant, even after controlling for household income, apart from highest attained education and having tested positive for COVID-19 at any time, which were no longer significant after controlling for household income.Respondents who reported being “stressed or worried about having enough food to meet my household’s basic needs as a result of the COVID-19 pandemic in the past two weeks” were more likely to report using a food bank compared to those who did not (12.6% vs 2.0%) [OR = 6.9 95% CI 4.6–10.1]. Similarly, those who reported sometimes  or often  worrying that food would run out before they got money to buy more in the last 12 months had significantly higher odds of reporting food bank use compared to those who did not. After controlling for the potentially confounding effects of household income, the magnitude of associations between food worry and food bank usage were diminished, but remained strong and statistically significant for those who reported being “stressed or worried about having enough food to meet my household’s basic needs as a result of the COVID-19 pandemic in the past two weeks” (yes versus no)  as well as for those who endorsed “sometimes true” or “often” true” (each versus “never true”)  for “worrying that food would run out before they got money to buy more in the last 12 months”. While respondents who reported food-related worry were more likely to report food bank use than those who did not across all models, less than one in four respondents who reported often worrying that food would run out used a food bank at any point since the onset of the pandemic.This study builds upon data collected in the early months of the COVID-19 pandemic , 28, 29,We cannot directly compare the current findings with rates of food-worry before the onset of the COVID-19 pandemic as one of the core food worry measures used here was novel and the other, which draws on a well-used survey item from the Household Food Security Survey Module, is seldom reported as a stand-alone estimate. However, these findings point to the potential value of monitoring and reporting food-related worry in its own right in future studies to understand the nuanced experiences that shape food insecurity and its consequences. We also note that national surveys using the HFSSM also typically ask about experiences over a 12-month reference period, and our first food worry estimate only referenced food worry in the past two weeks. Therefore, estimates generated here likely underestimated prevalence of episodic worries experienced across a longer timeframe in the early months of the pandemic.Our findings align with and further extend a robust body of scholarly evidence that has repeatedly found that Canada’s social safety net was insufficient for mitigating the food-related worries and financial consequences of the pandemic for many adults living in Canada , 39. In Our results align with evidence from Men & Tarasuk’s analysis of the Canadian Perspectives Survey Series 2 data, which reported that by May 2020, less than 10% of respondents had made use of charity-based food programs during the early stages of the pandemic . Food BaCanadian public discourse and legislative debates frequently focus on food charity and food banks specifically as a primary strategy to address food-access related challenges and insecurity –68. UponOur results build on existing Canadian evidence documenting many limitations of charitable food programming generated before the COVID-19 pandemic , 69, 70.This study illustrates that overall food bank usage remained relatively low during the pandemic in Canada, but there were notable socio-demographic and health-related characteristics that distinguished service users. For example, the odds of accessing food banks were significantly higher for younger adults, particularly those aged 18–34, and groups who were likely disproportionately impacted by labor market conditions and the economic fallout from the pandemic, particularly those with the lowest incomes . Raciali. Furthermore, while most food bank users report more severe and protracted forms of food insecurity, there is little evidence that longer duration or higher frequency of food bank usage substantively diminishes the severity or stress related to food insecurity for most users [These food bank usage characteristics align with prior research identifying that people who use food banks report more protracted experiences of severe poverty, financial vulnerability, food insecurity, precarious employment, and medical challenges –82. An ast users , 83–85. The findings from our nationally representative data are similar to those from other countries where large unmet gaps in food needs are concentrated among people who are marginalized and/or living in poverty. Inadequate social safety nets have been documented in several other high-income countries, with growing evidence that despite the proliferation and entrenchment of food banks as a primary response to unmet food need, charity-based food approaches have failed to address the growing challenges of food insecurity before or during the COVID-19 pandemic , 57–59. The sampling plan to facilitate this large sample size was designed to be nationally representative of the Canadian population by age, gender, region, and income  and faciFindings regarding the persistently high levels of food worry across survey rounds were robust to the use of two different measures of food-related worry. The first food worry item used in all four rounds of the survey was initially developed and used for pandemic research in the UK , to faciOther important limitations of this study’s design include the quantitative, cross-sectional nature from which the nuanced experiences of those experiencing food worry cannot be discerned. We are also unable to draw any specific conclusions here about how specific COVID-19 related policy changes or food program challenges buffered or exacerbated experiences of food worry or food program usage. For example, in the early months of the pandemic, strict physical distancing measures, and evolving public health recommendations likely shaped decisions to visit crowded spaces like food banks. Some Canadians  were also able to access $500 per week income supplements for up to 28 weeks from the Canadian Emergency Response Benefit (CERB) if they were unable to work because of the pandemic . While tThis paper provides timely insights regarding the prevalence of food-related worry and the socio-demographic characteristics of adults who accessed food banks in Canada in the context of the COVID-19 pandemic. Despite persistently high rates of food insecurity measured before and during the COVID-19 pandemic in Canada and the high rates of food-related worry reported here, relatively few adults accessed food banks or other charity-based community programs in 2020–2021. Adults facing social and structural barriers were more likely to report using a food bank during the pandemic, even after controlling for household income in statistical models, yet most food bank users still reported worry that food would run out before they got money to buy more.Findings align with previous research, which find that Canadian policies and programs during the pandemic were insufficient to buffer against food-related worry, particularly among those already experiencing multiple dimensions of structural inequities , 25. OurAdditional file 1.Additional file 2.


The current treatment of chronic HBV infection, pegylated interferon-α (pegIFNα) and nucleos(t)ide analog (NA), can suppress HBV replication, reverse liver inflammation and fibrosis and reduce the risks of cirrhosis, HCC, and HBV-related deaths, but relapse is common when the treatment is stopped before HBsAg loss. There have been major efforts to develop a cure for HBV, defined as sustained HBsAg loss after a finite course of therapy. This requires the suppression of HBV replication and viral protein production and the restoration of immune response to HBV. Direct-acting antivirals targeting virus entry, capsid assembly, viral protein production and secretion are in clinical trials. Immune modulatory therapies to stimulate adaptive or innate immunity and/or to remove immune blockade are being tested. NAs are included in most and pegIFNα in some regimens. Despite the combination of 2 or more therapies, HBsAg loss remains rare in part because HbsAg can be derived not only from the covalently closed circular DNA but also from the integrated HBV DNA. Achievement of a functional HBV cure will require therapies to eliminate or silence covalently closed circular DNA and integrated HBV DNA. In addition, assays to differentiate the source of circulating HBsAg and to determine HBV immune recovery, as well as standardization and improvement of assays for HBV RNA and hepatitis B core-related antigen, surrogate markers for covalently closed circular DNA transcription, are needed to accurately assess response and to target treatments according to patient/disease characteristics. Platform trials will allow the comparison of multiple combinations and channel patients with different characteristics to the treatment that is most likely to succeed. Safety is paramount, given the excellent safety profile of NA therapy.  Hepatitis B virus (HBV) infection remains a global health problem despite the availability of safe and effective vaccines for 4 decades, with an estimated 1.5 million new infections, 296 million chronically infected persons, and 820,000 HBV-related deaths in 2019.The challenges to HBV cure relate to the reservoirs for HBV replication and antigen production covalently closed circular DNA (cccDNA) and integrated HBV DNA and the impaired innate and adaptive immune responses against HBV. The episomal cccDNA, located in the hepatocyte nucleus, serves as a transcriptional template for all HBV RNAs, including pregenomic RNA (pgRNA), which is reverse transcribed into HBV DNA and messenger RNAs, which are translated into viral proteins Figure . cccDNA The error in primer translocation during HBV DNA replication may result in double-stranded linear DNA instead of relaxed circular DNA.Patients with chronic HBV infection have impaired innate and adaptive immune responses to HBV. Persistently high viral antigen levels had been incriminated to result in the exhaustion of HBV-specific T cellsAt the 2016 American Association for the Study of Liver Diseases (AASLD) and the European Association for the Study of the Liver (EASL) HBV Treatment Endpoints Conference,The definition of HBV functional cure adopted in 2016 and remained in place at the 2019 EASL-AASLD HBV Treatment Endpoint ConferenceIt should be noted that other reasons for choosing HBsAg loss as the anchor for functional HBV cure include the low risk of virologic relapse when treatment is stopped, given the desire for finite therapy, the potential for incremental clinical benefit compared with HBV DNA suppression without HBsAg loss,Low end-of-treatment HBsAg level is the best predictor of HBsAg loss after NA withdrawal and a strong predictor of HBsAg loss in ongoing trials of new HBV therapies.The complete suppression of cccDNA transcriptional activity was the original premise of the 2016 definition of HBV functional cure because serum HBsAg levels correlate better with hepatic cccDNA concentrations than serum HBV DNA levels. In the last 5 years, many studies have shown that serum HBV RNA and HBcrAg levels are better surrogate markers for hepatic cccDNA transcriptional activity than serum HBsAg levels.Partial cure is rarely achieved after the discontinuation of pegIFNα or NA therapy. In most patients who remain HBsAg positive, HBV DNA is detectable after stopping pegIFNα or NA though some patients may remain in clinical remission  <2 times the upper limit of normal). Whether it is possible to achieve partial cure with new therapies remains to be determined; however, caution must be exercised when treatment is stopped before HBsAg loss as severe flares, including hepatic decompensation, have been reported in clinical trials of new antiviral drugs.IFN exerts modest antiviral effects but it can enhance host innate and adaptive immune responses against HBV.NAs act primarily through inhibiting the reverse transcription of pgRNA to HBV DNA. Among the approved NAs, entecavir, tenofovir disoproxil fumarate (TDF), and tenofovir alafenamide are preferred because of their high antiviral potency and low risk of antiviral drug resistance. Despite DNA suppression, only 2%–5% of patients lost HBsAg after 10-year treatment.Recent studies showed that although viral relapse is universal when NAs are stopped, HBeAg-negative patients who discontinued NA after at least 2–4 years of complete HBV DNA suppression had higher rates of HBsAg loss with a cumulative incidence of 13%–41% by post-treatment year 5–6 compared with <5% in patients who continued NA.Although the Asian Pacific Association for the Study of the Liver and the EASL guidelines recommend that NAs may be stopped in selected HBeAg-negative patients who have completed >2–3 years of treatment with undetectable HBV DNA and agree to close follow-up,de novo combination of IFN and NA have not shown an improvement in HBsAg loss except for 1 study showing de novo combination of pegIFNα and TDF, resulted in a higher rate of HBsAg loss than pegIFNα or TDF monotherapy, but the benefit was mainly seen in patients with HBV genotype A.Most studies evaluating http://links.lww.com/HC9/A187).New drugs are needed to achieve functional HBV cure even if less ambitious endpoints are adopted; however, NAs will remain a backbone in suppressing HBV DNA replication, and pegIFNα may still play a role in decreasing HBsAg production or in facilitating HBsAg loss after it has been reduced to low levels. New therapies in development include direct-acting antivirals acting on different targets in the HBV life cycle, and immune modulators aimed to revive or stimulate HBV-specific immune response or to remove immune blockade Figures  and 2. MBulevirtide, a synthetic lipopeptide corresponding to the HBV preS1 region, blocks the entry of HBV into hepatocytes through the sodium taurocholate cotransporting polypeptide receptor, preventing infection of uninfected hepatocytes. Data on bulevirtide in chronic HBV monoinfection are limited. Studies in chronic HBV/HDV infection showed that bulevirtide alone had minimal effect on HBsAg decline with greater effect when used in combination with pegIFNα, but HBsAg loss is rare.Capsid assembly modulators (CAMs) can interfere with multiple steps in the HBV life cycle. CAMs currently in clinical trials primarily act by interfering with capsid formation resulting in aberrant or empty capsids, thereby decreasing HBV DNA replication. New CAMs aim to achieve higher intracellular concentrations that are necessary for interference with capsid disassembly (cccDNA establishment) and with intracellular recycling of capsids (cccDNA replenishment).Clinical trials of a combination of CAMs and NAs have shown an additive effect in decreasing HBV DNA and pgRNA levels but minimal effect on HBeAg and HBsAg levels suggesting that the main effect of CAM is interfering with pgRNA packaging and HBV DNA replication.Translation inhibitors, including siRNAs and antisense oligonucleotide (ASO) ASO, silence HBV RNA, thereby inhibiting HBV DNA replication and viral protein production.10 HBsAg decline after 3–4 doses lasting for 6–9 months post-treatment and 38% had sustained >1 log10 HBsAg reduction 11 months from last dosing.10 from pretreatment level) without ALT flares was observed after up to 44 weeks follow-up.Clinical trials of the N-acetylgalactosamine-conjugated siRNAs   showed 2–3 logA phase 2 trial of bepirovirsen, an unconjugated ASO targeting all HBV RNAs, showed a marked decrease in HBsAg level at week 24 in the highest dose (300 mg) group, with 26% and 29% having undetectable HBsAg at the end of treatment, and 9% and 10% having undetectable HBsAg as well as unquantifiable HBV DNA after 24-week post-treatment follow-up, in patients with versus without concurrent NA, respectively.The REEF-1 trial evaluated a triple combination of an siRNA (JNJ-3989), a CAM (JNJ-6379), and NA. Surprisingly, the triple combination resulted in less marked decrease in HBsAg level than the dual combination of siRNA and NA.Nucleic acid polymers (NAPs) and S-antigen traffic inhibiting oligonucleotide polymers can block the release of subviral particles from hepatocytes.Clinical trials of toll-like receptor 7 (TLR7) and TLR8 agonists showed minimal clinical efficacy despite increased cytokine production by T cells and NK cell activation.10 at 6 months) and 1 patient lost HBsAg lasting 8 months after the last dose.Previous attempts with therapeutic vaccines have not resulted in clinical efficacy.Other approaches, including autologous T-cell engineering with chimeric antigen receptor and T-cell receptor–redirected T cells, and soluble T-cell receptors linked to anti-CD3 antibodies (ImmTavs) are in preclinical or early phase clinical trials.Infusion of anti-HBs can neutralize circulating HBsAg and prevent the new infection of hepatocytes, but effects would be short lived unless HBsAg production is decreased and delivery of anti-HBs sustained. Several strategies to provide sustained delivery of anti-HBs, including the use of viral vectors, are being explored. Adding 12 weeks of a neutralizing monoclonal antibody VIR-3434 to VIR-2218 (siRNA) decreased HBsAg level to <10 IU/mL at the end of treatment in most patients though none achieved HBsAg loss.Checkpoint inhibitors such as programmed death receptor-1 blocking antibodies remove the blockade on T-cell function. A phase 1b study of 22 patients receiving 12 weeks of nivolumab (programmed death receptor-1 inhibitor), with or without GS-4774, a therapeutic T-cell vaccine, showed 3 (13.6%) patients in the high-dose arm achieved HBsAg reduction >0.5 log at week 24 and 1 patient (4.5%) had undetectable HBsAg lasting 12 months after therapy.Combination therapies will be needed to achieve a functional cure. To date, only the following combinations have resulted in off-treatment HBsAg loss: NA+NAP+pegIFNα, ASO+/−NA, siRNA+peg-IFNα. Although the highest rate of functional cure was reported with NA+NAP+pegIFNα, these results need to be confirmed in larger trials, and the high frequency of marked ALT flares remains a concern. The results of bepirovirsen +/−NA showed the most promise though only one third of those who lost HBsAg at the end of treatment remained HBsAg negative 24 weeks later, and longer follow-up will be needed to determine the durability of HBsAg loss. Of note, although the addition of NA did not seem to make a difference in HBeAg-negative patients, a combination with NA seemed necessary in HBeAg-positive patients. Other combination therapies that have resulted in end-of-treatment HBsAg loss include PD-L1 antibody+NA or siRNA+neutralizing monoclonal antibody, whether these responses will be sustained off-treatment remain to be determined. Of note, adding CAM to siRNA+NA did not confer any benefit in 2 trials, whereas adding pegIFNα to siRNA seemed to increase the odds of HBsAg loss during treatment though the durability of response is unknown.Functional HBV cure, defined as off-treatment sustained HBsAg loss, remains the goal of new HBV therapies. However, alternative endpoints may need to be adopted as intermediate steps to determine the success of clinical trials. The phase of clinical trials, the patient population studied, and the mode of action of the drugs tested will determine the choice of the most appropriate endpoints. At each AASLD-EASL HBV Treatment Endpoint Conference, representatives from the US Food and Drug Administration and the European Medicines Agency have expressed flexibility and encouraged industry sponsors to engage in consultative discussions when planning clinical trials. Achievement of any of the intermediate steps: decrease in HBsAg level to <100 IU/mL, complete silencing of cccDNA transcription, or partial cure after a finite course of treatment  in a high percentage of patients is a step forward compared with current therapies and are worthy goals in the foreseeable future.Achievement of a functional HBV cure will require the complete suppression of HBV DNA replication, inhibition of HBsAg production, and restoration/boosting of innate and adaptive immune responses against HBV Figure . It is eNAs will remain a backbone in suppressing HBV DNA replication. CAMs, entry inhibitors, and translation inhibitors can also contribute.Translation inhibitors, siRNA, and ASO will be pivotal in inhibiting HBsAg and other HBV protein production, allowing the recovery of the exhausted immune responses. PegIFNα can augment the inhibition of HBsAg production or facilitate HBsAg loss after HBsAg has been reduced to low levels. Other antivirals that may contribute to decreased HBsAg production include next-generation CAMs, entry inhibitors, and NAPs. Immune modulatory therapies such as therapeutic vaccines and checkpoint inhibitors can also contribute to inhibiting HBsAg production.To achieve a high rate of HBsAg loss, HBsAg production from integrated HBV DNA needs to be inhibited by eliminating integrated HBV DNA through gene editing with a risk of off-target effects or generation of undesired mutations or eliminating hepatocytes that harbor integrated HBV DNA with risk of severe flares. Alternatively, silencing of integrated HBV DNA transcription may be achieved through epigenetic modifications, which may be more specific and reversible. A major challenge is differentiating whether HBsAg that remains in circulation is derived from cccDNA or integrated HBV DNA. This distinction is critical if complete suppression of cccDNA transcription is the endpoint of treatment. A recent study proposed that this can be achieved using a digital droplet PCR assay targeting midportion or 3′ terminal end of HBV S transcripts, the latter being absent in transcripts derived from integrated HBV DNA, but this assay relies on liver tissue and an assay that can be applied to blood samples is needed.The complete suppression of cccDNA transcription is best determined by quantifying intrahepatic pgRNA, but this is not practical for clinical trials. Surrogate serum markers, including HBV RNA and HBcrAg, which have been shown to correlate with cccDNA transcription, may be used if standardized assays with improved sensitivity for these markers are available.Restoration of immune control is critical in sustaining HBsAg loss. Off-treatment reappearance of HBsAg in patients with undetectable HBsAg at the end of siRNA and ASO treatment trials has been attributed to lack of immune recovery. The availability of blood-based assays to characterize immune status would facilitate the identification of patients who need immunomodulatory therapies and the tailoring of these therapies to the immune phenotype of the patient. Although progress has been made, such assays/markers remain elusive.Most trials have focused on patients with active disease virally suppressed on NA because they met guidelines criteria for treatment, constitute the majority of patients regularly followed in hepatology clinics, and have a low risk of hepatitis flares. However, this is a heterogeneous population with varying durations of NA therapy, a wide range in HBsAg levels and immune reconstitution, and a generally unknown HBV genotype. Post hoc analysis of several trials showed high rates of HBsAg loss in patients with low baseline HBsAg levels; future trials might enrich for such patients and possibly include inactive carriers, though it would be important to demonstrate whether the investigational therapies accelerated HBsAg loss in patients who are already on the path to HBsAg loss. Recent studies showed that the duration and not the phase of HBV infection belie the degree of immune exhaustion,To date, most trials have used traditional designs testing one drug or one combination of drugs at a time. The efficiency can be improved using platform trials where multiple drugs can be tested using the same infrastructure, adding or removing new drugs based on initial results, and using the same controls for multiple comparisons. Platform trials can include patients with different characteristics channeled to the therapies that are most likely to succeed, enabling patients to be enrolled in the treatment arm that fit them best. Platform trials will require access to multiple drugs and collaborations among companies competing for the same goal.Efficient enrollment is key to success of clinical trials. A major hurdle in enrolling patients in clinical trials of new HBV therapies is safety. NAs are administered orally and have excellent safety profile. Although many patients on long-term NA therapy desire to have their HBV infection cured, they are rightfully concerned about the risks of experimental therapies and inconvenience  with uncertain benefit. Thus, stringent safety monitoring and detailed plans for managing adverse events, particularly hepatitis flares, and methods to differentiate potentially beneficial from harmful flares, must be in place. Although there is a strong desire for finite therapy, treatment withdrawal must be justified and closely monitored. Until recently, ALT is the focus of post-treatment safety monitoring and criteria for resuming treatment, but data from studies of NA withdrawal and new therapies have shown that a marked increase in HBV DNA levels often precede ALT flares and might be a safer trigger for reinitiating treatment.Sustained HBsAg loss can be accomplished in selected patients with current therapies, for example, pegIFNα in HBeAg-positive patients with genotype A, and NA withdrawal in White HBeAg-negative patients with HBsAg level <1000 IU/mL. HBsAg loss has also been observed in the clinical trials of some new therapies, for example, checkpoint inhibitor and NA, ASO with or without NA, NAP with NA and pegIFNα, predominantly in patients with low baseline HBsAg levels. Expanding the options for these “easy-to-cure” patients should be encouraged, but effort to find a cure for the majority left behind must continue. Clinical trials of drugs that have shown efficacy in decreasing cccDNA transcription or HBsAg production should continue, and the achievement of intermediate endpoints should propel further development, which may include collaboration among pharmaceutical companies and the design of platform trials comparing multiple drug combinations with the matching of patients to the best-suited treatment.Although antiviral approaches alone may result in sustained HBsAg loss in some patients, a functional HBV cure will likely require immunomodulatory therapies in many patients. The development of reliable immunologic assays or markers to identify which patients need immunomodulatory therapies and how to tailor the choice of immunomodulatory therapy to maximize the likelihood of HBsAg loss is urgently needed. Similarly, the availability of reliable blood-based assays that can determine the source of circulating HBsAg is needed to select therapies directed at the right target and to assess response.Although the previous goal of 30% sustained HBsAg loss after 1–2 years of treatment might seem more remote now than in 2016, the efforts invested in the last 5 years have provided important tools and insights toward achieving that goal. Stakeholders, including regulatory agencies, pharmaceutical industry, scientists, clinicians, and patients, should promote collaborations and conduct of platform trials with treatment arms tailored according to patient characteristics to safely achieve functional HBV cure in incremental steps.


Sébastien Bélanger and colleagues  demonstrate that plastid transformation can also be used to generate traits outside of the organelle's function.Plastids are a group of plant organelles that carry out specific biosynthetic and metabolic functions. All plastids differentiate from proplastids into one of several specialized organelles. These include chloroplasts (the site of photosynthesis), chromoplasts , and amyloplasts (which store starch in root cells). Recent advances in plastid transformation technology have shown numerous advantages and provide an alternative approach to nuclear genome transformation. For instance, the presence of multiple copies of the plastid genome offers high transgene expression, the lack of heterochromatin in the plastid results in no transgene silencing, and the maternal inheritance of the plastid genome reduces the likelihood of transgene contamination by pollen . In theiPhytoene Desaturase (PDS) gene into the tobacco plastid genome. The first transgene expressed a partial PDS cDNA sequence from 2 convergent plastid promoters and consequently produced double-stranded RNA (dsRNA line). The other transgenes contained a partial PDS cDNA sequence in either sense or antisense orientation, driven by a plastid promoter to express sense RNA or antisense RNA (sense line and antisense line). The pigment gene PDS was chosen to monitor gene silencing as its dysfunction leads to a bleaching phenotype in normally green tissues. The antisense line did not exhibit any phenotype, whereas both the dsRNA and sense lines displayed bleaching phenotypes, indicating that PDS expression was repressed. The maternal inheritance of plastid transgenic traits was confirmed through reciprocal crosses.To test the ability of a plastid-encoded DNA fragment to silence nuclear genes, the authors transformed transgenes corresponding to the nuclear PDS genes, PDS1 and PDS2, that share 99% sequence identity. Both PDS1 and PDS2 mRNAs showed significant reduction in the dsRNA line compared with the wild-type line, suggesting the bleached phenotype of the dsRNA line is a consequence of PDS mRNA knockdown. At a later developmental stage, pigment-deficient plants begin to lose their bleaching phenotype while the PDS mRNAs remain reduced. This indicates that the reversion of the bleaching phenotype is regulated by additional mechanisms independent of PDS transcript levels. Conversely, although the sense line had a similar pigment-deficient phenotype, no changes in PDS mRNA accumulation were observed, indicating a different mechanism such as translational-level regulation is involved in the sense line and has yet to be investigated.The tobacco nuclear genome encodes 2 PDS sequence in the dsRNA line. Further analyses revealed that the majority of small RNAs on both RNA strands were 21-nt, and these small interfering RNAs (siRNAs) are generated in a phased manner, similar to the plant-specific class of phased secondary siRNAs (phasiRNAs). Because phasiRNAs are triggered by a microRNA and generated through iterative cleavage of dsRNA by Dicer-Like proteins (PDS phasiRNAs or if these are possibly “triggerless.”High-resolution small RNA sequencing of seedlings with transformed plastids revealed abundant that matched the proteins , Dicer pThe next question raised was whether a nuclear-encoded complementary RNA is required for siRNA processing. To address this, the authors assessed small RNAs in 2 additional tobacco lines to express dsRNA against insect gene targets that are not present in the tobacco genome. One line expressed dsRNA using 2 convergent plastid promoters, while the other line expressed a hairpin dsRNA with a single promoter. In both cases, siRNAs were abundant and mapped to both RNA strands. Additionally, 21-nt phasiRNAs were the predominant siRNA species. The above results indicate the plastid-expressed dsRNA does not need the complementary nuclear-encoded RNAs to produce siRNAs.This study shows that plastid-driven transgene expression can lead to the production of siRNAs that target nuclear-encoded mRNAs. The dsRNA likely enters the cytoplasm from the plastid and generates 21-nt phasiRNAs for gene silencing .. This f