Recent findings show that TEs recruit epigenetic control on the expression of effector genes as part of the coordinated infection strategy. In addition to genome plasticity and diversity, fungal pathogenicity is an area of economic concern. A survey of TE distribution suggests that their proximity to pathogenicity genes TEs may act as sites for emergence of novel pathogenicity factors via nucleotide changes and expansion or reduction of the gene family. Through a systematic survey of literature, we were able to conclude that the role of TEs in fungi is wide: ranging from genome plasticity, pathogenicity to adaptive behavior in evolution. This review also identifies the gaps in knowledge that requires further elucidation for a better understanding of TEs’ contribution to genome architecture and versatility.Transposable elements (TEs) are agents of genetic variability in phytopathogens as they are a source of adaptive evolution through genome diversification. Although many studies have uncovered information on TEs, the exact mechanism behind TE-induced changes within the genome remains poorly understood. Furthermore, convergent trends towards bigger genomes, emergence of novel genes and gain or loss of genes implicate a TE-regulated genome plasticity of fungal phytopathogens. TEs are able to alter gene expression by revamping the  Sustainable production of crops is crucial to safeguard food security and maintain socioeconomic stability. However, crop production is constantly challenged by biotic and abiotic stresses that threaten sustainable food production. Diseases such as blast, (rice), stem rust (wheat) and corn smut (maize), are main contributors to yield losses in many agriculturally dependent countries . Major pUnderstanding fungal pathogen lifestyles and mechanisms of evolution is crucial in controlling the spread of fungal diseases. One sure way of understanding the organism is by unravelling the regulatory mechanisms underlying the molecular composition of the fungi. This will provide us with the understanding of the adaptive nature of these organisms, their mechanism of survival and pathogenicity. The advent of the genomic era has resulted in a surge in the availability of microbial genome sequences, including that of the destructive fungal phytopathogens . ThroughA brief overview on TEs will provide the information on the roles played by these elements in genomes. Since the discovery of TEs in the mid-1940s , perceptThe first TE classification system was proposed by Finnegan  in 1989 In Wicker’s proposal, TEs are hierarchically classified into class, subclass, order, superfamily, family and subfamily . SimilarClass II TEs transpose via DNA intermediate and are divided into subclass I (“cut-and-paste”) and subclass II (“copy-and-paste”). Subclass I TEs are divided into two orders namely terminal inverted repeats (TIR) and Crypton whereas subclass II TEs are divided into Helitron and Maverick orders  Figure . TranspoGenome plasticity enables organisms to adapt to environmental changes and occupy novel niches. Adaptive evolution mediated by TEs is facilitated by recombination events resulting in genomic diversification. This is achieved through genomic changes, which persist under positive selection in fungal pathogens. TEs contribute towards adaptive genetic variation through: (1) TE insertion into coding genes , (2) TE Möller and Stukenbrock, (2017) on the other hand described fungal phytopathogen evolution as managed versus natural ecosystem and ‘trench warfare’ versus ‘arm race’. The observed trend is that fungi have evolved rapidly and are highly adaptive to new environments. However, TE density does not directly correlate to a rapid rate of evolution as genomes with low TE content can exhibit rapid evolution . ThroughMagnaporthe oryzae through PacBio sequencing allows for scrutiny of a lineage-specific [Verticillium dahliae, a broad-range fungal plant pathogen causing vascular wilt disease. This region is rich in active TEs and also harbors effector genes, including Ave1 that is significant for host recognition in initiating virulence [Blumeria graminis f. sp hordei (Bgh), a powdery mildews fungus that employs a particularly high-speed, one-speed genome. TEs that are evenly distributed in the Bgh genome are mostly associated with coding regions of various biological processes including avirulence genes. TEs occupy most of the Bgh genome and the genome is gene poor but TE-rich throughout [Furthermore, TEs’ remarkable role in adaptive evolution can be elucidated through the two-speed genome concept of pathogen evolution . This cospecific  region tspecific  as obserirulence . Furtherirulence . Howeverroughout .Fusarium oxysporum adaptation to a new host is facilitated by TE-rich insertions in the ‘B’ chromosomes [Alternaria sp [M. oryzae is a consequence of host shift in Setaria millet, following the opportunistic timeline of rice domestication. Host shift is often promoted in new species that are highly important, widely distributed and with high-density population such as rice. The host shift is founded on the expansion of TE copy number, MGR586 (Pot3), followed by the loss of a specific avirulence gene, AVR-co39, that allows for the jump to new host. The loss of avirulence gene indicates that the AVR-R gene interaction may prevent the initiation process of host shift hence resulting in subsequent inability to invade rice. Hence, TEs have a big influence on host specialization by promoting the loss of avirulence genes and the breakdown of complete resistance, based on rare-specific R genes [TEs’ mediated impact on the fungal pathogen adaptability to new environment and overcoming host resistance is presented in the following examples. Ma et al. (2010) reported that omosomes . Broadennaria sp . TE-medinaria sp . For ins R genes ,55.M. oryzae on 6 Avr-genes indicate the significant presence or absence in polymorphism and a high ratio of non-synonymous to synonymous substitution which may be the underlying mechanism used to evade the host plant system through rapid adaptive evolution. Interestingly, repeat sequences including TEs are found flanking most of these reported Avr-genes [M. oryzae and F. oxysporum reported mutations in their repetitive genome compartments [Verticillium dahliae and F. oxysporum, we stipulate that recombination may contribute towards adaptive evolution in a primarily clonal community. This observation is further substantiated by reports that M. oryzae and Puccinia striiformis formae speciales (f. sp.) tritici have evolved towards generation of asexual clonal linages with ferocious spreading abilities. In addition, high TE activity in Aspergillus oryzae [M. oryzae [A study involving 62 strains of vr-genes . Furtherartments ,58,59. Fs oryzae  and M. o. oryzae  is hypotTherefore, to maintain a pathogenic lifestyle, adaptation to host and environment is essential. We hypothesize ecological adaptation to host and environment may be achieved through the combinatorial and mutational traits of TE. The TE-induced speciation may promote the emergence of advantageous alleles that are essential in host-pathogen evolution against changing environments, host and fungicide. We believe that if the pathogenicity clusters are maintained under the pressure of TE evolutionary adaptations, the pathogen is then likely to be dynamic in resistance, and adaptation to the current host or new hosts. Nevertheless, host fitness is diminished through non-adaptive and deleterious changes caused by TEs. TEs’ role in adaptive evolution requires further studies in population genetics and extensive analysis of fungal genomes to connect the effect of TEs to mutational fitness.Genome plasticity can be described as alterations observed in a genome structure, which can be characterized by changes in genome organization, chromosome number and genome size . Genome Blumeria graminis DH14) and the basidiomycete rust fungi (45% repetitive of 220 Mbp in Melampsora lini CH5) [Hemileia vastatrix, is 797 Mbp which is bigger than the 710 Mbp size of its diploid Coffea canephora host plant [Ustilago maydis for example exhibited compacted genome and lack of LTR retrotransposon content [TEs promote genome size expansion due to its replicative nature where convergence towards a large genome is an observed trend for fungal phytopathogens . Even thini CH5) . The genst plant ,64. Thisst plant . These sst plant . Howeverst plant . The gen content . M. oryzae infecting different hosts [Oryza Ina168, a M. oryzae isolate infecting rice, in comparison with other isolates that infect foxtail, goosegrass, wheat and oat. This observation coincides with the event of host shift and host specialization where some genes were lost through purifying selection and genetic drift [Blumeria graminis f.sp. hordei, an obligate biotrophic powdery mildew fungus with 64% of its genome made up of TEs. The expansion in retrotransposition activities predominantly from non-LTR groups and the inactivation of repeat-induced point mutations (RIPs) in the obligate biotroph not only increased its genome size but also led to gene losses compared to autotrophic ascomycetes. These missing genes are related to primary and secondary metabolism, plant cell wall depolymerization and toxin secretion which are pathogenesis-associated functions not required in an obligate biotrophic fungus [M. oryzae and B. graminis, we can deduce that TEs not only orchestrates the host specialization of fungal phytopathogens but also influence the adaptability to changing environments.TEs’ activities are also significantly linked to the gain or loss of genes where the gain/loss in polymorphism can be observed in the genomes of nt hosts . For insic drift . The samcis-regulatory elements can emerge from TEs that have undergone point mutations [cis-regulatory sequences in TEs to mimic the host promoters. For example, in the LTR of mammalian endogenous retrovirus [cis-regulatory sequences and RNA Polymerase II promoters [Another effect from TE insertion is the restructuring of transcriptional and post-transcriptional regulatory networks . TE inseutations . Autonomtrovirus ,70, the romoters . Throughromoters ,60. Throromoters  and enviromoters . As DNA romoters .M. oryzae where genes that encode secreted proteins are found within 1 kb flanking distance from TEs [M. oryzae Y34 and P131, against the reference genome of M. oryzae 70-15. Lack of conserved TE loci between these isolates was observed despite thousands that were found, thus supporting active transposition activities. Notably, most TEs found are in close proximity with duplicated genes and isolate-specific genes, including avirulence genes. The reported duplicated genes with known functions are predicted to be associated with primary and secondary metabolism and host interaction, which is similar to the observations made in isolate 70-15. The transposition of TEs and its association with duplicated genes as well as isolate-specific genes may be the most critical factor in contributing to genome variation in M. oryzae [Effector proteins that are secreted by fungal pathogens to promote colonization interfere with host defense and result in necrosis. By the same token, effector proteins can be recognized by their complementary plant resistance genes, leading to the activation of defense responses in plants . Pathogefrom TEs . Active . oryzae .Mycosphaerella fijiensis, a fungus that causes black Sigatoka in Banana has 11.7% of TEs in its genome. A total of 339 protein encoding genes including crucial metabolism and pathogenicity-associated genes were found within 1000 bp upstream from these TEs [Mycosphaerella graminicola, a fungal wheat pathogen that causes Septoria tritici blotch, contains approximately 17% repeat elements, mostly Class I TEs (70%). A few genes involved in pathogenicity were in proximity with these nested clusters of TEs. These repeat clusters may act as a ‘breeding ground’ where new pathogenicity determinants can emerge as shown previously in studies of fungi and oomycetes [U. maydis, Sporisorium reilianum, Ustilago hordei and Sporisorium scitamineum, genes encoding secreted effectors are found pooled in clusters. Notably, these clusters are in the vicinity of repeats, particularly uncharacterized repeats in comparison with other genes, suggesting the need to further characterize these repeats and their contribution to the genome [The TE-pathogenicity gene association is also displayed in other fungal pathogens. hese TEs   and thence expand on its host range. TE mediated inactivation or deletion of PAMP-encoding genes or effector genes important for host recognition results in gain of virulence  by evadiis locus .F. oxysporum. TEs are over-represented in the lineage-specific region of F. oxysporum (broad host range) with over 74% of its genomic TEs present in the LS regions, an observation not seen in both F. verticillioides (narrow host range) and F. graminearium (narrow host range). LS region is presumed acquired through horizontal transfer and mobilization of host-specific unique chromosomes into this region. The transfer of these LS chromosomes has been experimentally proven to change non-pathogenic strains to pathogenic strains. In addition, the differences in repetitive sequence content between Fusarium species suggest that TEs introduce pathogenicity to new environments by providing genes required for host-specificity [Rhizoctonia solani is an area that is worth further studies. The comparative analyses between different AGs namely AG8, AG3 and AG1-1A, highlights differences in SNP and repeat content which may reflect the differences seen in their host range and infection strategies [Variable inter- or intra- species TE abundance can also be potentially linked with a gain of virulence and subsequent expansion of host range. Ma et al., (2010) highlighted the gain of virulence through the presence of mobile pathogenicity chromosomes and its correlation with enriched TE content in LS regions of cificity . Furtherrategies   between TE abundance and the spectrum of host range  in a phytopathogenic fungi?Are TE activities and its presence random in nature or preferentially inserted to alter the genome dynamics in order to suit the needs for genome survival?Can TEs be manipulated to provide translational applications in mitigating crop disease?A deeper look into the effects of TEs in agriculturally relevant traits such as pathogenicity, host range, and genome plasticity will be useful to provide molecular perspectives on the driving factors of virulence to hosts. Nonetheless, there are still unexplored gaps in the study of TEs that leaves room for more interpretational analyses pertaining to TE traits and the possible impact on phytopathogens as follows:Through this review we note that there is no specific trend seen within fungi from the same family or even genus. However, with improving technologies, annotation mechanisms, and the outpouring of genome data, more information may be surmised on the TE’s contribution to host-pathogen interaction and this information may be of potential use in addressing plant diseases. Through in depth studies of population genetics and genome databases, a clearer picture may be obtained on the roles of these elements in fungal phytopathogens and their contribution in their respective lifestyles.


A girl born at a gestational age of 36 weeks presented with cyanosis. Echocardiography led to the diagnosis of right isomerism, a complete atrioventricular septal defect, pulmonary atresia, an aortic valve arising from the right ventricle and a total abnormal pulmonary venous drainage (TAPVD) of the infracardiac type, with a collector vein connecting to a liver vein, draining into the left-sided atrium. After venous umbilical catheter placement, alprostadil was started, whereafter saturation increased to 85–90%. Computed tomography (CT) Fig. , performAwareness of the possibility of an abnormal position of the umbilical venous catheter in the presence of an infracardiac TAPVD is warranted since catheter-related endothelial damage and thrombus formation could lead to stenosis of the pulmonary collector vein prompting urgent intervention.


Cocaine- and amphetamine-regulated transcript (CART) is widely expressed in the hypothalamus and an important regulator of energy homeostasis; however, the specific contributions of different CART neuronal populations to this process are not known. Here, we show that depolarization of mouse arcuate nucleus (Arc) CART neurons via DREADD technology decreases energy expenditure and physical activity, while it exerts the opposite effects in CART neurons in the lateral hypothalamus (LHA). Importantly, when stimulating these neuronal populations in the absence of CART, the effects were attenuated. In contrast, while activation of CART neurons in the LHA stimulated feeding in the presence of CART, endogenous CART inhibited food intake in response to Arc CART neuron activation. Taken together, these results demonstrate anorexigenic but anabolic effects of CART upon Arc neuron activation, and orexigenic but catabolic effects upon LHA-neuron activation, highlighting the complex and nuclei-specific functions of CART in controlling feeding and energy homeostasis. Cart prepropeptide (Cartpt) and involved in the regulation of a diverse range of physiological functions including food intake and energy homeostasis, thermoregulation, reward as well as stress processing (Cartpt-knockout (KO) mice and the effects of intracerebroventricular (i.c.v.) injection of CART peptides suggest anorexigenic and catabolic actions of CART (The neuropeptide cocaine- and amphetamine-regulated transcript (CART) is encoded by ocessing . While t of CART . In cont of CART , indicatCartpt with the orexigenic melanin-concentrating hormone  and glutamate decarboxylase  in the LHA  and the lateral hypothalamic area (LHA) show high expression of CART and play a central role in a variety of homeostatic functions, including the regulation of energy homeostasis . The Arc the LHA , support the LHA . Importa the LHA , highligCre-recombinase-dependent adeno-associated viral (AAV) vector to specific hypothalamic nuclei of Cartpt-cre knock-in mice. Comparison of the metabolic responses of cre/creCartpt mice (which do not express CART) and cre/+Cartpt mice provides specific insights into the direct contribution of CART to the effects of CART neuron activation at these two hypothalamic nuclei.In this study, we aimed to investigate the contribution of CART neuron activation to the regulation of energy expenditure focusing on the Arc and LHA, respectively. Activation of CART neurons was achieved by the use of the designer receptors exclusively activated by designer drugs (DREADD) technology  delivereN-oxide (CNO) defined by c-Fos expression were determined. Fluorescence microscopy confirmed that in cre/+Cartpt mice stereotaxically injected with AAV-hM3Dq-mCherry into the Arc . Parameters of energy expenditure, physical activity and respiratory exchange ratio (RER) under free-feeding conditions were measured using open-circuit indirect calorimetry. The depolarization of the Arc CART neurons was achieved by i.p. injection of CNO and the same mice were used as their own control injected with saline 24 hr later.In order to investigate the contribution of Arc CART to energy expenditure regulation, we unilaterally injected 10-week-old CART-expressing cre/creCartpt mice  were also investigated in cre/creCartpt mice where CART is absent when compared to the same mice injected with saline   , contrase/+ mice . The elee/+ mice , highligcre/creCartpt mice, which lack CART, had no significant effect on physical activity  when physical activity in mice is naturally lower . Resultsing CART . Similaring CART , that ising CART .cre/creCartpt mice  demonstrates that the presence of CART promotes the increase in both BAT and lumbar temperatures  are at play for the control of body temperature by endogenous CART in this neuronal population.An increase in body temperatures could be due to increased thermogenesis or reduced heat loss, or both. Since the tail is a key regulator of heat dissipation in rodents, and a decrease in tail skin temperature is indicative of decreased heat loss, which could contribute to an increase in body temperature  we also e/+ mice , indicate/+ mice . Howevercre mice , suggestcre/+Cartpt mice at 1 and 3 hr after CNO injection was greater than that in cre/creCartpt mice . Food and water intake as well as body weight were assessed for up to 6 hr after CNO injection, the time period where most of the responses to this activation occur .cre/creCartpt mice 26Sortm9(EGFP/Rpl10a)Amc/J mice in which the GFP-tagged ribosomal protein (GFP-L10a) is expressed upon Cre-recombinase activation, and we refer to this new line as CART-TRAP mice in which the GFP-L10a ribosomal protein is solely produced in Cartpt-expressing neurons (Cartpt transcript in the immunoprecipitated (IP) RNA relative to the corresponding unprocessed RNA (Input) sample (Pmch), orexin (Hcrt), GABAergic markers , neurotensin (Nts), the leptin receptor (Lepr) and galanin . Interestingly, we observed higher expression of most of the markers of the study in the IP population relative to the input sample . For this, we crossed our  neurons . Consist) sample . Using a) sample  that aret sample , except cre/creCartpt mice that are devoid of endogenous CART. Furthermore, while food intake is not affected by activation of intact Arc CART neurons, activation of these neurons in the absence of CART leads to an increase in feeding, suggesting an inhibitory role of CART in these neurons by opposing the orexigenic action of other co-expressing neurotransmitter(s). In contrast, activation of LHA CART neurons results in a marked increase in energy expenditure, locomotion, body temperature and feeding. Although these effects induced by LHA CART neuronal activation can be achieved regardless of the presence or absence of CART, the presence of CART allows these responses to occur under lower level of activation  and at a greater magnitude than in the absence of CART, indicating that CART is the predominant neurotransmitter responsible for these effects.The present study demonstrates the critical and distinct roles of CART neurons in the Arc and LHA in the control of feeding behavior and energy homeostasis, and more importantly represents the first report to dissect the specific contribution of CART to these processes. Activation of Arc CART neurons leads to significant decreases in energy expenditure, locomotion and body temperature, and these effects are solely due to the contribution of CART since they are absent in Arc→hM3Dq Cartpt-/- mice display increased body weight gain and adipose mass  . Interes obesity . Similarecreased .Cartpt expression at thermo-neutral conditions as compared to room temperature  , but not for vesicular GABA release (Slc32a1) in both MCH and orexin neurons  , anothere (Hcrt)  showing  neurons . The gre neurons . Importa neurons , consist neurons .Taken together, our study demonstrates the differential metabolic effects of endogenous CART in response to pharmacogenetic activation of neurons of the Arc or LHA. The findings from this study reinforce CART’s multifaceted context-dependent and region-specific functions in the modulation of feeding, thermoregulation and energy balance. Interestingly, apparent dissociation between the effects of CART on food intake and energy expenditure was unraveled in response to neuronal activation. Specifically, the presence of CART blunted the otherwise increase in food intake and body weight gain in response to Arc→hM3Dq CART neuron activation, while at the same time decreasing locomotion and energy expenditure. Opposite effects were observed in response to LHA→hM3Dq CART neuron activation, where the presence of CART promoted food intake and ingestive behavior, while increasing locomotion and energy expenditure. From a behavioral perspective, it could also be inferred that endogenous CART promotes a state of behavioral inhibition in response to Arc→hM3Dq CART neuron activation, while promoting behavioral activation in response to LHA→hM3Dq CART neuron activation.All experimental and animal care procedures were approved by the Garvan Institute/St. Vincent’s Hospital Animal Ethics Committee and were conducted in agreement with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Male mice were used for all experiments and were housed under conditions of controlled temperature (22°C) and illumination . Mice were provided with ad libitum access to water and a standard chow diet .Cartpt-cre knock-in mouse line on a C57/Bl6 background as described previously  recombinase-recombination target (FRT)-flanked neomycin selection cassette. A cassette carrying the tamoxifen-inducible Cre-recombinase gene followed by an internal ribosome entry site (IRES) that drives the expression of the mCherry red fluorescence reporter gene was inserted. LoxP sites were placed immediately upstream of the initiation codon of the Cartpt and Cre genes respectively, which allows the replacement of the Cartpt gene by Cre. Positively targeted ES cell clones were injected into blastocysts. Chimeric offspring were crossed to create heterozygous conditional CART mice with the floxed gene (lox/+Cartpt), which were then bred with a germline Flp-recombinase mouse line to remove the neomycin cassette and further crossed to homozygosity to generate the conditional CART floxed knockout line (lox/loxCartpt). The lox/loxCartpt line was then crossed with C57BL/6J-Tg(Zp3-cre)82Knw/KnwJ mice that expressed Cre specifically in the oocytes, resulting in a rearrangement that combines the inducible Cre gene with the endogenous CART promoter. Subsequent crossing to homozygosity ultimately generated the novel inducible Cartpt-cre knock-in mouse line (cre/creCartpt). Since the endogenous Cartpt gene has been replaced by the Cre-recombinase gene, the resultant cre/creCartpt mice also represent a classical germline CART knockout mouse line (−/−Cartpt), while the heterozygous cre/+Cartpt mice do express endogenous CART. To activate the CART promoter controlled Cre-recombinase gene 1 μl of the active metabolite of tamoxifen, 4-hydroxytamoxifen (4-OHT) (20 mg/mL)  dissolved in absolute ethanol was injected into the left lateral cerebral ventricle (LV) of 10–12 week-old cre/creCartpt and cre/+Cartpt mice. The injection coordinates relative to Bregma were –0.34 mm anteroposterior,+1.0 mm mediolateral, and –2.5 mm dorsoventral corresponding to the left LV.To investigate the effects of activation of CART expressing neurons, CART neuron-specific introduction of genetic elements in an adult-onset inducible manner was enabled through the generation of a conditional eviously . In shorCre-recombinase 1 μl of the active metabolite of tamoxifen, 4-OHT (20 mg/mL)  was injected into the LV two weeks before the isolation of the LHA for TRAP experiments.For CART-TRAP mice, to activate the 13 viral particles/mL, Addgene #44361 Bryan Roth) in an inducible manner. Upon exposure to tamoxifen, Cre-recombinase in crecre and Cartptcre/+Cartpt mice is translocated to the nucleus of CART neurons, thereby catalyzing the inversion of the loxP-flanked cassette resulting in hM3Dq receptor expression in CART neurons only. Accordingly, activation of the hM3Dq-expressing CART neurons was triggered remotely by i.p. injection of CNO (0.3 mg/kg).Chemogenetic activation of CART neurons was performed by injection of a rAAV6-hSyn-DIO-hM3Dq-mCherry vector  was injected at the right Arc . LHA hM3Dq receptor expression was achieved by either bilateral or unilateral injection at the LHA . To activate the Cre-recombinase for mediating the transgene inversion and expression, 1 μl of the active metabolite of tamoxifen, 4-hydroxytamoxifen (4-OHT) (20 mg/mL)  dissolved in absolute ethanol was then injected unilaterally into the left lateral cerebral ventricle (LV). The injection coordinates relative to Bregma were –0.34 mm anteroposterior,+1.0 mm mediolateral, and –2.5 mm dorsoventral corresponding to the left LV. Mice were allowed two weeks of recovery with regular body weight assessment prior to any phenotypic characterization procedures.Adult-onset central CART neuron-specific expression of the hM3Dq receptor in the Arc or LHA was achieved by intra-hypothalamic injection of the hM3Dq-mCherry viral vector in parallel with i.c.v. injection of 1 μl of the active metabolite of tamoxifen, 4-hydroxytamoxifen (4-OHT) (20 mg/mL)  in eviously . 0.75 μlcre/creCartpt or cre/+Cartpt and LHA→hM3Dq cre/creCartpt or cre/+Cartpt mice. At least 6 mice were included for each of the groups resulting in a number of 6–12 mice per site of injection and genotype.For simplicity, the post-surgery subjects are referred to as Arc→hM3Dq cre/creCartpt and cre/+Cartpt mice (n ≥ 6 for each of 6 groups) in response to treatment of either CNO or saline control was measured. After a 2–3 weeks of recovery period after AAV-hSyn-DIO-hM3Dq-mCherry delivery, all mice were at 15 wks of age and fed a standard laboratory chow. All mice undergoing unilateral hM3Dq delivery were assessed for basal feeding behavior for 24 hr as described previously . Food intake was measured 24 hr later at 5:00 pm, during which the same experiment was repeated immediately in a crossover design, such that mice receiving saline treatment before were i.p. injected with CNO, and vice versa. The crossover design allows each mouse to serve as both the experimental and the control subject, such internal biological control method minimizes the sample size required for achieving statistical strength.TM, Sable Systems International, NV USA). This calorimetry system consists of 8 metabolic cages  each equipped with water bottles and food hoppers connected to load cells for food and water intake monitoring, and all animals had ad libitum access to standard rodent chow and water throughout the study  were evaluated for metabolic parameters and physical activity at 17–18 wks of age. For energy metabolism, metabolic rate of the unilateral injected cohorts was measured by indirect calorimetry using an 8-chamber open-circuit calorimeter   . At 5:00TM, Sable Systems International, NV USA)  . As desccre/creCartpt and cre/+Cartpt cohorts by non-invasive high-sensitivity infrared imaging with a high-sensitivity infrared camera  as described previously (Whole body skin temperature and skin temperature at the interscapular brown adipose tissue (BAT) were measured for both the eviously . TemperaCartpt-cre knock-in mice as described  diluted 1:1500 in PBST with 2% normal goat serum and 1% bovine serum albumin. Following three 10 min washes in PBST, sections were incubated with anti-rabbit Alexa Fluor 488  for 2 hr .For visualization of Fos immunoreactivity at the sites of AAV-hM3Dq delivery, hypothalamic brain sections containing the targeted Arc or LHA regions from respective Arc→hM3Dq and LHA→hM3Dq mice were investigated using a Zeiss Axiophot microscope equipped with a ProgRes 3008 digital camera (Carl Zeiss).In order to investigate co-expressed transmitters within the hM3Dq-expressing CART neurons immunohistochemistry was also performed for GAD67 , secondary anti-mouse Alexa Fluor 488 antibody , MCH , secondary anti-rabbit Alexa Fluor 488 antibody  and orexin , secondary anti-goat Alexa Fluor 488 antibody .Cartpt, Gad1, Gad2, Gal, Hcrt, Lepr, Nts, Pmch, Slc32a1 as indicated in Supplementary fActb and expressed as relative to input values. Sequences of the oligonucleotide primers used for these PCRs can be found in TRAP experiment was performed based on the previously published protocol with some modifications . ReagentAll data are presented as means ± SEM. Differences amongst mouse groups of various genotypes and treatments were assessed by ANOVA or repeated-measures ANOVA combined with Bonferroni post-hoc analysis where appropriate. Energy expenditure, RER and physical activity over the continuous 24 hr period were averaged for the whole 24 hr period, as well as the 12 hr light and dark phases individually. Statistical analyses were performed with GraphPad Prism 6 for Mac OS X  and SPSS for Mac OS X version 16.0.1 . Statistical significance was defined as p value≤0.05. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The following individual involved in review of your submission has agreed to reveal his identity: Michael A Cowley (Reviewer #3).Thank you for submitting your article "Opposing effects of arcuate nucleus and lateral hypothalamic CART neurons on energy expenditure" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:Farzi and colleagues took advantage of the chemogenetic DREADD technology to generate original results, which further delineate the function of hypothalamic CART and CART neurons in food intake and energy expenditure. They report opposite effects following depolarization of Arc CART versus LHA CART, with Arc CART neurons decreasing, while LHA CART increasing, energy expenditure and physical activity, effects that are attenuated in the absence of CART. They also show different effects on food intake following activation of those CART populations. They conclude that activation of Arc CART neurons produces anorexigenic but catabolic effects, while activation of LHA CART produces orexigenic but catabolic effects. Overall, the manuscript reads well and presents novel results clarifying CART neurons' role in energy metabolism. However, the reviewer enthusiasm is limited by the fact that the study is still descriptive in nature and is at some extent confusing to follow.Essential revisions:Figure 1 is not conveying the point they are trying to make. The authors could add additional rostro-caudal pictures allowing a better reflection of the infection pattern. Moreover, the pictures show a very lateral injection pattern, especially for the Arc. The c-Fos photomicrographs are not very convincing and could be improved.Injecting mice with empty vectors is not the only control needed. An additional group of WT receiving DREADD viruses and CNO would be welcome.cre/+Cartptmice – I assume this means that they had some CART?) reduced energy expenditure, skin temperature over intrascapular BAT depots, and physical activity. The BAT temperature data suggest that there may have been changes in sympathetic nervous system outflow. In contrast, the effect of neuronal activation on food intake and body weight was not seen in the cre/+Cartptmice, but was seen in the cre/creCartptmice – the effect was a reduction in food intake.Activation of Arc CART neurons  treatment (with CNO in the drinking water) in, for instance the LHA model, would have possibly been a nice addition. The LHA model that allowed for bilateral depolarizations was apparently the most interesting one and could have been further exploited.Neuroanatomical experiments to further describe the identity of CART neurons (particularly in the LHA) would have added useful information to determine whether the LHA CART neurons are part of circuitries genuinely involved in energy homeostasis regulation.cre/+Cartptmice increased energy expenditure and physical activity, but did not alter dermal temperature over the scapulae. It also increased food intake. The effect appeared to be stronger in mice with bilateral injection of the activator construct, which is not surprising.Activation of CART neurons in the LHA of  Essential revisions:Figure 1 is not conveying the point they are trying to make. The authors could add additional rostro-caudal pictures allowing a better reflection of the infection pattern. Moreover, the pictures show a very lateral injection pattern, especially for the Arc. The c-Fos photomicrographs are not very convincing and could be improved.We have expanded this analysis and provide now additional picture that more clearly demonstrate the points we like to convey. These figures include rostro-caudal pictures reflecting the infection pattern of the Arc and LHA injection sites  as well as improved c-Fos micrographs showing CNO-induced c-Fos expression in hM3Dq-mCherry-positive CART neurons. The expression pattern are in agreement with our previous in situ analysis (https://doi.org/10.1016/j.npep.2016.03.006) and also reflects our cautious approach to aim our injections on the fringe of the Arc to let the viral vector to diffuse into it rather than causing major tissue damage through the needle track. Moreover, the significant alterations seen in the different physiological readouts in the injected versus the controls are testimony for the accuracy of our viral delivery.Injecting mice with empty vectors is not the only control needed. An additional group of WT receiving DREADD viruses and CNO would be welcome.We fully agree and actually these controls were already included in our original manuscript . These negative controls are now described in more detail in the Results section and the lack of responses in WT mice receiving DREADD viruses and CNO are presented in Figure 2—figure supplement 1, Figure 9—figure supplement 1 and Figure 10—figure supplement 1.cre/+ mice – I assume this means that they had some CART?) reduced energy expenditure, skin temperature over intrascapular BAT depots, and physical activity. The BAT temperature data suggest that there may have been changes in sympathetic nervous system outflow. In contrast the effect of neuronal activation on food intake and body weight was not seen in the Cartptcre/+ mice, but was seen in the Cartptcre/cre mice – the effect was a reduction in food intake.Activation of Arc CART neurons  temperature covering the inter-scapular brown adipose tissue (BAT) as a measure of BAT thermogenesis. In fact, temperature differences between the inter-scapular and lumbar areas from the same animal (TBAT-TBack) are a better measure of the thermogenic contribution of inter-scapular brown adipose tissue. For that reason, we now also present this TBAT-TBack temperature difference in response to Arc CART neuron activation . As there is no significant difference between the changes in surface temperature of the BAT and lumbar regions, we conclude that the observed decrease in surface (skin) temperature is not due to a specific change in BAT thermogenic activity. Furthermore, the observation that the decrease in temperature is paralleled by a decrease in physical activity suggests that decreased activity related thermogenesis may at least in part contribute to the observed temperature changes. This aspect is now also addressed in the Results section.cre/cre. This issue makes it very hard to interpret the data. This should be made clearer in a revision.The mouse models description is very confusing: what germline CART KO was used? What dose of tamoxifen and when was it injected? What oocyte expressing-Cre line did authors use? Was it a ZP3-cre? Tamoxifen-inducible? Information about the source and background should be included. Was icv tam or hydroxyl tam used? The gene nomenclature does not follow the guidelines of the journal, by example, CART-cre should be CartptWe have expanded the description of the different models in the Materials and methods section and include additional citations describing the original generation of the different models. In short, all mice are on a C57Bl/6 background. To generate the CART Cre knock-in line, we crossed our mice with a floxed CART gene that downstream also contained an inducible Cre-recombinase cassette with a germline oozyte (ZP3-Cre) line resulting in the removal of the CART coding sequence and its simultaneous replacement with the downstream inducible Cre-recombinase gene, which now is under the control of the endogenous CART promoter. The germline CART KO mice were generated by crossing these heterozygous CART Cre knock-in mice to homozygousity. To activate the CART promoter controlled Cre-recombinase gene the active hydroxyl tamoxifen was injected icv.Apparently, the energy balance measurements were only acutely performed, which limits the speculation on the effects of CART neurons in the regulation of energy balance. A sub-chronic (14 days) treatment (with CNO in the drinking water) in, for instance the LHA model, would have possibly been a nice addition. The LHA model that allowed for bilateral depolarizations was apparently the most interesting one and could have been further exploited.eLife  and in other journals . While the potential chronic treatment with CNO is a possible addition we believe this would not add significant additional information to the already extensive evaluation of these models.Our study was designed according to standard procedures and technologies used and published previously in Neuroanatomical experiments to further describe the identity of CART neurons (particularly in the LHA) would have added useful information to determine whether the LHA CART neurons are part of circuitries genuinely involved in energy homeostasis regulation.cre/+Cartptmice to generate the cre/+;TRAPlox/loxCART mice (CART-TRAP), where the ribosomal L10a-GFP fusion protein is only produced in CART neurons .To further characterise the nature of the LHA CART neurons we employed IHC colocalisation experiments as well as ribosomal affinity purification technology (TRAP). For this, GFP-L10a fusion protein encoding Rosa26CAG-EGFP (TRAP) mice were crossed onto our Cartpt transcript in the immunoprecipitated (IP) RNA relative to the corresponding unprocessed RNA (Input) sample . Using a candidate approach, we tested for the enrichment of known markers that are involved in energy homeostasis control including MCH and galanin, GABAergic markers and neurotensin.qPCR performed on mRNA isolated by immunoprecipitation with an anti-GFP antibody against the GFP-tagged ribosomal unit from LHA preparations of these mice confirmed a significant enrichment of Interestingly, there was overlap with all of these other neuropeptides except galanin, suggesting that CART neurons in the LH are highly heterogeneous and co-localise with various neuropeptides within the LHA. This great heterogeneity in CART neurons could be one of the reasons for the great spectrum of functional output from these LHA CART neurons.cre/+ mice increased energy expenditure and physical activity, but did not alter dermal temperature over the scapulae. It also increased food intake. The effect appeared to be stronger in mice with bilateral injection of the activator construct, which is not surprising.Activation of CART neurons in the LHA of CartptWhile it may not be surprising that the bilateral activation shows a stronger effect it actually is consistent with the observed heterogeneity of the CART neurons in the LH where probably only a subset of them contributes to this effect and this maybe on the threshold when they are only unilaterally activated.Furthermore, the fact that specific activation of LHA CART neurons in the presence of endogenous CART increases EE but does not alter BAT surface temperature suggests that this may be driven by the increase in physical activity and the fuel used for this is directly sourced from the increased food intake.


Post-cardiac injury syndrome (PCIS) as a delayed complication of permanent pacemaker implantation has rarely been reported in the literature. A 67-year-old man who recently underwent a dual chamber permanent pacemaker implantation came to the hospital for increasing dyspnea and chest discomfort. A diagnosis of pericarditis was made, and the patient was discharged on ibuprofen therapy. He presented to our facility a month later with worsening dyspnea and chest discomfort despite recommended therapy. A computerized tomography (CT) scan of the chest revealed a large right-sided pleural effusion, requiring chest tube placement and drainage. A pleural fluid analysis revealed exudative effusion with elevated pH. The pleural fluid analysis was negative for infectious etiology. A perforation of the atrial wall was considered given the proximity of the atrial pacer lead and overlying pericardial effusion. However, no conclusive evidence of cardiac chamber perforation was found on echocardiogram or CT scan. A pacemaker interrogation was normal. A repeat CT scan showed the resolution of pleural effusion, and the chest tube was discontinued. A possible explanation for the absence of predominant pericardial findings may be the previous use of non-steroidal anti-inflammatory therapy. Post-cardiac injury syndrome (PCIS) is a heterogeneous group of disorders occurring in response to a variety of cardiac injuries, including cardiac surgery, blunt chest wall trauma, and coronary and endovascular interventions, among others. PCIS can occur following cardiac device placement, and only a limited number of cases have been reported in the literature. We present a case of delayed PCIS after permanent pacemaker insertion presenting as a large unilateral pleural effusion. A cardiac perforation was also considered in the differential diagnosis because of the proximity of the pacemaker leads to the atrial wall and the associated pleuro-pericardial effusion, causing diagnostic and therapeutic challenges.A 67-year-old non-smoker man with a past medical history of non-obstructive coronary artery disease, hyperlipidemia, essential hypertension, paroxysmal atrial fibrillation, and subclinical hypothyroidism and no prior history of autoimmune disease in the family underwent permanent dual chamber pacemaker implantation for sinus node dysfunction. The patient was discharged home without any immediate procedural complications but returned to the hospital two weeks later with increasing dyspnea and chest discomfort. He had extensive testing, including workup for ischemic heart disease. A left heart catheterization was done, revealing non-obstructive coronary artery disease. A transthoracic echocardiogram revealed a small pericardial effusion without any other echocardiographic abnormalities. He was discharged home on ibuprofen with a diagnosis of pericarditis. He was admitted to our facility a month later with worsening dyspnea and non-productive cough. He denied fever, chills, or chest pain at presentation. The physical examination was consistent with decreased breath sounds in the right middle and lower lung fields. Chest radiograph Figure  and compPost-cardiac injury syndrome (PCIS) is an inflammatory process involving the pleura and pericardium in response to cardiac injury. PCIS can occur in response to endocardial, myocardial, or epicardial injury and has been described after percutaneous coronary interventions. PCIS is infrequently reported in association with pacemaker device insertion or radiofrequency ablation with only a few case reports in the literature -3. PCIS The spectrum of PCIS ranges from pericarditis with pericardial effusion to massive pleuropericardial effusions and cardiac tamponade. Pericardial effusion with or without the features of tamponade was found in the majority of cases . PCIS maIt is important to differentiate PCIS from pacemaker lead perforation to prevent unnecessary diagnostic and therapeutic interventions. Lead perforation may cause a change in pacing and sensing parameters on pacemaker interrogation. However, PCIS is less likely to cause such changes. Concomitant anticoagulation, female gender, and steroid use increases the risk of lead perforation but don’t affect the risk of developing PCIS . InsteadPleuropulmonary manifestations of PCIS have been reported but rarely with permanent pacemaker insertion. The pleural fluid analysis reported exudative effusions in the majority of cases. It may be difficult to differentiate the exudative effusion seen with PCIS from an infectious cause especially early in the course of the illness. A high pleural fluid pH may favor the diagnosis of PCIS over infectious causes, as seen in our patient . A largeInvestigations for pacemaker-associated PCIS should include device interrogation, chest radiography, transthoracic or transesophageal echocardiography, computerized tomography (CT) of the chest, and laboratory studies, including complete blood counts, and inflammatory markers, including erythrocyte sedimentation rate (ESR). Some investigators have suggested a role of anti-heart antibodies in the diagnosis; however, their clinical significance remains unclear . A CT scThis case is unusual because of the development of a large pleural effusion despite being treated with recommended therapy and the absence of predominant pericardial symptoms. A possible explanation for the absence of predominant pericardial findings may be the previous use of non-steroidal anti-inflammatory medication. Clinical, radiographic, and laboratory features are helpful in differentiating between PCIS and lead perforation. A pleural fluid analysis showing a high pH is helpful in the diagnosis of PCIS. A chest CT scan may be helpful in cases with an unclear diagnosis, but this may fail to provide a clear diagnosis in some cases. A delayed cardiac perforation from pacemaker leads is a life-threatening condition that can mimic PCIS and should always be considered. A high pleural fluid pH helps differentiate an effusion due to PCIS from one caused by a pulmonary infectious process that is usually associated with low pH.


The immune system represents a compelling example of evolution in action: antibody diversity is created by a variety of molecular mechanisms, and then selection acts to preserve and propagate the most useful antibodies. We have combined immune repertoire sequencing with population genetics to measure the strength of selection on various antibody lineages in humans who have been vaccinated for influenza. Antibodies are created and refined by somatic evolution in B cell populations, which endows the human immune system with the ability to recognize and eliminate diverse pathogens. However, the evolutionary processes that sculpt antibody repertoires remain poorly understood. Here, using an unbiased repertoire-scale approach, we show that the population genetic signatures of evolution are evident in human B cell lineages and reveal how antibodies evolve somatically. We measured the dynamics and genetic diversity of B cell responses in five adults longitudinally before and after influenza vaccination using high-throughput antibody repertoire sequencing. We identified vaccine-responsive B cell lineages that carry signatures of selective sweeps driven by positive selection, and discovered that they often display evidence for selective sweeps favoring multiple subclones. We also found persistent B cell lineages that exhibit stable population dynamics and carry signatures of neutral drift. By exploiting the relationship between B cell fitness and antibody binding affinity, we demonstrate the potential for using phylogenetic approaches to identify antibodies with high binding affinity. This quantitative characterization reveals that antibody repertoires are shaped by an unexpectedly broad spectrum of evolutionary processes and shows how signatures of evolutionary history can be harnessed for antibody discovery and engineering. Antibodies are created through evolutionary processes involving mutation and selection, all of which unfold in B cell populations. As proposed by Burnet in his “clonal selection theory” in 1957, the concepts of population genetics offer an avenue for understanding how antibody repertoires evolve . This selection drove recurrent selective sweeps during somatic evolution, in which antibody variants repeatedly arose via mutation and selectively expanded to become dominant within the clonal population. Many vaccine-responsive B cell lineages display evidence for selective sweeps favoring multiple subclones. Other abundant B cell lineages exhibit stable population dynamics and lack a response to vaccination; we show that these lineages carry signatures of neutral evolution . Finally, we present an approach for using phylogenetic information to identify potential high-affinity antibodies and affinity-enhancing mutations. Our results offer a detailed portrait of the somatic evolutionary processes that shape human antibody repertoires and link models of evolution with quantitative measurements of the human immune system.Here, we characterize the dynamics and somatic evolution of human B cell lineages using high-throughput sequencing of the antibody repertoire and analytical methods inspired by population genetics. We performed time-resolved measurements of antibody repertoires in healthy young adults before and after seasonal influenza vaccination. We identified vaccine-responsive B cell lineages that expanded dramatically after vaccination, and we show that patterns of genetic variation within these lineages reflect a history of strong positive selection (IGH) using RNA extracted from peripheral blood mononuclear cells . In contrast, across a similar time span in the absence of vaccination (between D0 and D5), only 6 ± 4 lineages within each subject expanded to this extent , which may be attributable to exposure to environmental antigens. Because most of these “vaccine-responsive” lineages were undetectable before vaccination, the identification of vaccine-responsive lineages was robust to the specific choice of fold-change (FC) cutoff . Together, these vaccine-responsive lineages accounted for 22 ± 12%  of each subject’s repertoire during peak response at D7. Vaccine-responsive antibodies have high levels of somatic mutation . We concluded that influenza vaccination triggers rapid recall of dozens of clonal B cell lineages in healthy human adults.To visualize how the composition of the antibody repertoire changed after vaccination, we examined the fractional abundance of clonal B cell lineages over time B. We defmutation E and aremutation F and G, SI Appendix, Table S1). Persistent lineages displayed remarkably stable population dynamics compared with vaccine-responsive lineages , implying balance in cellular turnover and mRNA expression levels. Persistent antibodies have low levels of somatic mutation . IGHJ4 was used in the vast majority of persistent lineages (86%), unlike the lineages in the rest of the repertoire . Thus, many human antibody repertoires possess a large complement of persistent B cell lineages, which have stable population dynamics on timescales of weeks and do not respond dynamically to influenza vaccination.We discovered that each subject harbored a distinct set of clonal B cell lineages that exhibited high abundance throughout the study and were unresponsive to vaccination . In continuously adapting asexual populations, the SFS exhibits a distinct excess of high-frequency variants, which can be used to rule out neutral models and infer positive selection . We compared the observed SFSs against population genetic models of neutral evolution with constant population size [Kingman coalescent .To quantitatively characterize the evolutionary histories of clonal B cell lineages, we examined the frequency spectrum of derived somatic mutations, also known as the site frequency spectrum (SFS). The SFS carries detailed information about evolutionary history and can be useful for detecting selective processes from snapshot sampling of genetic diversity . To quantitatively detect selection, we used Fay and Wu’s . Similarly, 43% of vaccine-responsive lineages deviate significantly from the neutral model with population expansion (P < 0.05). We also directly measured the nonmonotonicity of the SFS and found that 14% of vaccine-responsive lineages deviated significantly from neutrality by this alternative metric for selection . Nearly every subject had at least one vaccine-responsive lineage that evidently experienced selection .Next, we sought to characterize the patterns of somatic evolution at the resolution of individual clonal B cell lineages. While individual lineages have fewer somatic mutations and thus exhibit sparse spectra compared with population averages, we found that many vaccine-responsive lineages have a large excess of high-frequency mutations . Indeed, selection was detected at a rate that is consistent with a model in which every vaccine-responsive lineage evolved under strong positive selection , suggesting that the sensitivity of the statistical test, given the sizes of the sampled populations, accounted for the failure to detect selection in every vaccine-responsive lineage. In support of this, the signature of selection, as measured by the significance of Fay and Wu’s H compared with size-matched neutrally evolving lineages, showed a trend toward inverse correlation with the number of sampled sequences in the lineage . In turn, the number of sequences in the lineage correlated strongly with the total amount of nucleotide diversity , suggesting that reliable detection of selection relies on having sufficient mutational diversity to support phylogenetic analysis.The failure to detect selection in every vaccine-responsive lineage is consistent with statistical limits of detection arising from the population sizes of the lineages , which form the structural scaffold of the antibody molecule and typically evolve under purifying selection (High-frequency derived mutations are enriched within complementarity-determining regions (CDRs), which form the antibody-antigen binding interface and often evolve under positive selection , 15. Sucelection , 15. TogSI Appendix, Fig. S3A) as reflected in their balanced phylogenies, which are consistent with the absence of selection and characteristic of neutral drift-like evolution . Similarly, 88% of persistent lineages had no significant deviation from the neutral model with population expansion (P > 0.05). We also found no significant departure from neutrality for nearly every persistent lineage (99%) using the nonmonotonicity of the SFS as a metric for selection . Persistent lineages had large population sizes comparable to those of vaccine-responsive lineages , indicating that limits of detection arising from population size cannot explain the failure to detect selection. Indeed, the rate at which we detected selection on persistent lineages was much lower than the detection limit . Thus, persistent lineages evolve in a manner consistent with neutrality, suggesting that neutral birth-death processes are responsible for the expansion and maintenance of a substantial fraction of the human antibody repertoire.Persistent antibody lineages have a strikingly different mode of evolution. When we visualized the SFS as an average over all persistent lineages, we found its shape to be consistent with neutral evolution, lacking an excess of high-frequency somatic mutations D. Indeedvolution B. Using The molecular features of persistent lineages are characteristic of B-1 cells, a B cell subtype that has a different life history than the better-studied B-2 cells. Both persistent lineages and B-1 cells are mostly IgM , with a P < 10−9). Lineages with significant evidence of positive selection  expand more after vaccination than lineages without such evidence . Furthermore, regardless of the choice of FC cutoff for defining clonal expansion, many more positively selected lineages than nonpositively selected lineages undergo clonal expansion  but more than the FC cutoff for persistent lineages (2-fold), yielding a total of 450 lineages. We found that positive selection is highly correlated with clonal expansion . We found that 24% of vaccine-responsive lineages composed of >1,000 sequences harbor multiple subclones that have evidence of positive selection . In comparison with synonymous mutations, nonsynonymous fitness-enhancing mutations were highly enriched in CDRs  and depleted in FWRs  with the sole exception of FWR2 (P = 0.87). Thus, phylogenetic inference of fitness enhancement-associated mutations is consistent with the expected distribution of nonsynonymous and synonymous mutations in the tree based on the structural basis of antibody-antigen interactions (P < 8 × 10−11), consistent with the idea that mutations in CDRs, especially CDR3, can sometimes harm fitness because they disrupt antibody-antigen binding interfaces, suggesting that the traditional notion of purifying selection being confined to FWRs is overly simplistic. While these predictions must be validated experimentally via expression of antibodies with native heavy and light chain pairing, our results suggest that phylogenetic methods can reveal information about antibody affinity which is encoded in sequence diversity and potentially can be used to rapidly identify high-affinity antibodies and affinity-enhancing mutations.In line with a history of selective sweeps, phylogenetic inference revealed wide variation in fitness among sequences within vaccine-responsive B cell lineages, with some sequences predicted to have much higher fitness than other sequences in the same clonal lineage A. We ideractions –37. ThisIn summary, our results demonstrate that human antibody repertoires are shaped by a broad spectrum of somatic evolutionary processes. Prior efforts to detect selection in antibody genes focused on regions or residues in aggregate across many clonal B cell lineages –16, and On the other hand, persistent lineages display signatures of neutral drift-like evolution, revealing that nonselective processes generate a substantial fraction of human antibody repertoires and requiring that the conventional notion that selective processes are ubiquitous in antibody maturation be modified. This diversity of evolutionary modes likely reflects the diversity of life histories among distinct B cell types. The presence of large clonal lineages lacking molecular signatures of selection also provides an inherent control and constitutes evidence that the detection of such signatures in vaccine-responsive lineages is not an artifact of our approach, including a failure to correctly determine the germline sequence. In addition, the presence of large clonally expanded persistent lineages evidently displaying signatures of neutral drift indicates that population expansion itself cannot account for the signatures of selection observed in vaccine-responsive lineages. Importantly, our results suggest that the molecular signatures of selection distinguish vaccine-responsive lineages from other clonal lineages that are also highly abundant after vaccination. We have shown that molecular signatures of selection can be harnessed through phylogenetic approaches to identify sequences that were most favored by selection during affinity maturation and therefore likely encode high-affinity antibodies with potential utility for biomedical applications. High-throughput sequencing of human antibody repertoires and analysis through the lens of population genetics thus offer a promising avenue for antibody discovery and engineering.IGH constant region primers for reverse transcription and IGH variable region primers for second-strand cDNA synthesis followed by PCR, following Vollmers et al.  and then compared with simulations of evolutionary models using betatree , immediately before vaccination (D0), and 1, 4, 7, 9, and 11 days afterward . Peripheral blood mononuclear cells were isolated, total RNA was extracted, and sequencing libraries were prepared from 500 ng of total RNA using isotype-specific s et al.  and Horn IgBLAST , and isobetatree  or custobetatree , and phybetatree . Selectibetatree  of subtrbetatree .Supplementary File


Amplification of viral genomes is key to successful infection and spread. Herpes simplex virus 1 (HSV-1) is a successful and ubiquitous human virus. HSV-1 acute infection outcomes range from asymptomatic infections to life-threatening encephalitis. Primary infection leads to life-long, persistent, latent infection, which can periodically reactivate to result in characteristic “cold sores.” Herpesviruses are considered ancient viruses that have coevolved with their hosts for a long time, generating complex virus–host interactions [There are three phases of HSV-1 viral gene expression from the double-stranded DNA genome: immediate early (IE), early (E), and late (L). These are temporally and spatially regulated within the infected cell nucleus. IE proteins are involved in the takeover of gene transcription and countering of intrinsic host defenses. E proteins are mainly involved in viral DNA replication. L genes are activated after the onset of viral DNA replication, and their protein products are involved in assembly and packaging of progeny virions. Although this cascade of gene expression has been well characterized at the population level, the temporal variation among individual infected cells is less well understood, and it is unclear whether all entering genomes follow the entire viral genetic cascade.Sequestering of viral replication machinery at specific intracellular locations is a common phenomenon observed in virology. This highlights the important evolutionary benefits of viral-induced structures, which serve to both concentrate required proteins and also limit access by detrimental host factors. Like many other viruses, herpesviruses confine their gene expression and replication to distinct intranuclear sites known as replication compartments (RCs). In this short review, we discuss the formation of these structures, their ability to attract viral and cellular proteins, and the impact of interactions between distinct individual RCs.The HSV-1 genomes are packaged inside the viral capsid as a linear double-stranded DNA molecule that contains nicks and gaps . These gMany viral and host proteins are thought to assemble on viral genomes to form RCs. The RCs formed during HSV-1 infection were originally identified as nuclear sites containing viral DNA associated with viral single-stranded DNA–binding protein ICP8 and viral transcription activator ICP4 , 13. RCsThe nature of the viral DNA presumably contributes to protein recruitment. Viral DNA enters the nucleus as an open-ended molecule with nicks and gaps . These fRecent studies of subcellular compartments suggest a role for liquid phase separation as a way to sequester specific proteins within compartments that are not separated by membranes . These sViral RCs are dynamic structures that change during infection with respect to their size, position in the nucleus, and protein composition. As replication proceeds, the size of the RC grows , and theSeveral observations support the possibility that viral proteins encoded in one RC can spread to all other RCs within the same nucleus. First, complementation between coinfecting herpesviruses (even between different viruses in the family) is well established. Furthermore, in PRV we found that L capsid proteins from different genomes are distributed evenly among the newly formed capsids . This miBy detecting different viral genomes within a cell, we found that each genome maintains its own territory even at points of intersection between RCs , 7. ThisImaging RCs has revealed that, within single cells, not all RCs are the same size and shape . At earlThe distinct intranuclear structures formed during HSV-1 infection represent hubs for viral gene expression, replication, and encapsidation. Biochemical and imaging approaches have defined components of these viral-induced structures. Although these studies have helped to identify potential players in the process of viral replication, these findings raise many new questions about how these interactions impact the outcome of infection. Is there a competition between RCs for limited cellular resources? To what extent do viral RCs possess characteristics of liquid organelles? What is the significance of heterogeneity among RCs? Ongoing technology improvements will allow better understanding of the processes required for viral replication. Viral research has provided insights into many fundamental host mechanisms. Because viral replication is a rapid event that dramatically alters nuclear architecture, understanding mechanisms involved in RC formation, maintenance, and interactions will shed further light on structural processes and regulation within the mammalian nucleus.


Refractory vasodilation and catecholamine resistance are common in septic shock. Changes in receptor signaling, excessive production of nitric oxide, and absolute or relative deficiencies of vasoactive hormones, including cortisol, vasopressin, and angiotensin II, play a role. Angiotensin II (Ang II) was previously available as a vasopressor but removed from the market in the 1990s. Interest was re-ignited following the Angiotensin II for the Treatment of Vasodilatory Shock ATHOS-3) study, a randomized controlled trial in patients with refractory shock which confirmed that Ang II was effective at maintaining mean arterial pressure and reducing norepinephrine requirements without an increase in side effects . Recent  study, aThe major physiological effects of Ang II relate to maintenance of hemodynamic stability and fluid and electrolyte regulation Table . AngioteWe report the successful management of seven patients  with severe cardiorespiratory failure and refractory shock treated with extracorporeal membrane oxygenation (ECMO) who received Ang II in the context of the ATHOS-3 trial  or a comIn conclusion, in patients with severe cardio-respiratory failure requiring ECMO, treatment with Ang II in addition to standard supportive care enabled rapid decatecholaminization. Underlying ACE deficiency may be a contributing factor. Further studies are necessary to confirm the findings.


Hemodialysis (HD) remains a principal modality for renal replacement therapy worldwidefor patients with end stage kidney disease (ESKD) and a functioning dialysis vascularaccess is critical to providing adequate therapy. Despite all the technological advancesin the delivery of HD, the process of creating a vascular access has remained vastlyunchanged. Almost six decades since the creation of an autogenous fistula, the dialysiscommunity continues to struggle with creating a dependable method to remove blood fromthe body for HD therapyList: Barriers for timely creation of an arteriovenous fistulaSystem-related barriers:Late referral to a nephrologist by primary care physicians;Late referral to surgeons for AV access creation bynephrologists;Backlog with surgeons - clinic and operating room appointments;Lack of defined process for follow up visits after the surgery;Poor understanding of timely intervention for a primary non-maturingAVF.Patient-related barriers:Denial phase, non-compliance;Lack of education;Aging population;Multiple comorbidities;Socio-economic factors, such as transportation, family support,co-payments etc.Variable provider expertise:Inadequately trained nephrologists, interventionalists andsurgeons;Minimal importance to vascular access in training curricula;Inadequate emphasis on vessel preservation in advanced CKD populationamongst all healthcare providers;Inadequately trained allied health providers;Limited training resources.III-defined barriers:Poorly understood pathophysiology of AVF dysfunction;Hemodynamic impact of AVF.Siga E et al. have focused on the importance of a multidisciplinary access team inclusiveof a dedicated surgeon towards creating a successful AVFMany lessons have been learned over the past two decades since the implementation of FFBIand National Kidney Foundation-Kidney Disease Outcomes Quality Initiatives (NKF-KDOQI)Vascular Access Guidelines that can guide our future management goals.The first objective should focus on building a multi-disciplinary team consisting of alead nephrologist, a skilled and dedicated surgeon, an interventionalist, engageddialysis staff, a vascular access coordinator, and pre-dialysis counselor to helpmotivate and educate the patient. When possible, vascular access-focused education andplanning should begin early enough to avoid/minimize the need to use CVC. KDIGO 2006guidelines recommend that AVF should be placed at least 6 months before the anticipatedstart of HD treatments. Some authors have proposed placement of AVF at GFR ≤20mL/min/1.73 m6The second objective should focus on identifying a non-maturing AVF within six to eightweeks of placement in order to plan for early intervention and assist with thematuration process. The ultimate goal being minimizing the CVC exposure time andpreserving the central veins. This goal can be achieved if the caregivers and patientsare trained with proper physical examination technique to identify a problematic AVF. Ingeneral, mature AVF should have an access flow of 600 mL/min, be less than 0.6 cm belowthe surface of the skin, and have the minimal diameter of 0.6 cm (Rule of 6s). The FFBIteam has created several tools that can help improve the competency skills of the entiredialysis communityThe last objective should focus on strategies to improve long-term patency of afunctioning AVF. Several hurdles have been identified in achieving this goal and thebiggest barrier of all is breaking “old habits”. Long-term AVF patency can be improvedif collectively, as dialysis caregivers, we implement a fundamental change in ourpractice patterns. All well-functioning AVFs invariably become dysfunctional over time.Long-term patency of AVFs can be achieved by improving the overall process of care.Several key steps must be taken to ensure the functionality of the access over anextended period of time. These steps include:,Assessment and proper cannulation technique - Inadequately trained dialysispersonnel can cause major trauma to the vessel, often irreversible. Frequentstaff training and competency assessment for aseptic technique, correctneedle size, proper cannulation technique, and needle anchoring procedureare fundamental for maintaining patency8Early identification of dysfunction - History and physical examination toassess the presence of stenosis, thrombosis, aneurysms, and recognizingprolonged bleeding times and access pressures, which should be recognizedbefore and after each treatment for timely intervention. Using monitoringand surveillance data, such as ultrasound, access blood flow and pressure,are complimentary evidence to identify dysfunctional AVFs. Timelyrecognition of dysfunction can lead to timely referral for intervention.Identifying complications - Recognizing signs of infection, monitoring foraneurysms, symptoms of distal limb ischemia, and impact on cardiachemodynamics are relevant for providing optimal careEmpowering the patient - Every attempt should be made to educate and empowerthe patients to participate in their care. Engaged and involved patients canidentify certain problems before the complication becomes a major issue. Anengaged patient may be able to identify a change in the character of thethrill within an AVF before a thrombotic event. Success from earlyendovascular intervention in a poorly flowing AVF is higher than in acompletely thrombosed accessTraining the next generation - Nephrology curriculums across the globe placelittle emphasis on dialysis vascular access. Lessons learned from thecurrent generation will improve the quality of care for the next generationof patients. The worldwide epidemic of ESKD and the economic impact ofdialysis therapy on society warrants a major shift in the trainingcurriculum for all caregivers. Innovative research and better understandingof the changes in vascular biology following AVF creation may offer betteroptions for care in the future.


Primary testicular lymphoma is a rare testicular neoplasm that mainly affects elderly patients, with Human Immunodeficiency Virus (HIV) being a known risk factor in the younger population. Approximately 20% of patients will have disseminated disease with extra-nodal involvement at clinical presentation. Rarely, direct spread along the spermatic cord and gonadal vessels can occur and has been described in the literature. We present two cases of this phenomenon where the primary testicular tumour has spread along the gonadal vein to its origin at the inferior vena cava. Primary Testicular Lymphoma (PTL) is an uncommon neoplasm representing less than 5% of all testicular tumours and between 1-2% of all cases of non-Hodgkin's Lymphoma (NHL) , 4. AlonPTL typically presents as a unilateral painless testicular mass or scrotal swelling and is difficult to differentiate clinically and on imaging from germ cell tumours. Rarely pain may occur, while constitutional symptoms are indicative of systemic disease, which is present in 20% of patients at the time of diagnosis.NHL of the testis may be associated with extra-nodal involvement of the central nervous system (CNS), Waldeyer's ring, lungs, pleura, skin, and soft tissues as well as the contralateral testis. A small number of case reports however describe direct spread along the spermatic cord and gonadal vessels into the retroperitoneum . In the A previously well 77-year-old gentleman presented with a 6-week history of right-sided testicular swelling and gradual onset of pain with no preceding history of trauma or known malignancy. He was initially treated in primary care for suspected orchitis but due to persisting symptoms he was referred for a scrotal ultrasound.The ultrasound study  demonstrCT demonstrated an enhancing right-sided testicular mass  with sofFollowing a multidisciplinary team (MDT) discussion and with lymphoma being the main differential due to the distribution of the lesions, the testicular mass was biopsied under ultrasound guidance and histology results demonstrated diffuse large B-cell lymphoma . Lymphomatous tonsillar involvement was confirmed on biopsy and gastroscopy following an episode of haematemesis showed gastric infiltration, not evident on imaging. Imaging investigations were completed with whole spine and brain Magnetic Resonance Imaging (MRI) to assess for CNS involvement. The patient was subsequently commenced on chemotherapy for stage VI Diffuse large B-cell lymphoma, with follow-up 3-month imaging showing very good partial response.An 82-year-old patient with a background of monoclonal gammopathy of undetermined significance (MGUS) and previous prostate cancer treated with external beam radiation therapy presented with constitutional symptoms. On clinical examination an enlarged right testicle was noted and serum biochemistry revealed hypercalcaemia.A CT of the chest, abdomen, and pelvis was performed to assess for a new underlying malignancy or prostate cancer recurrence. The study demonstrated a large right-sided scrotal mass Figures  with sofA subsequent ultrasound  was perfAfter discussion at the urology cancer MDT a differential diagnosis of sarcoma and lymphoma was suggested and a decision for ultrasound guided biopsy of the testicular lesion was made, rather than orchiectomy due to epididymal and spermatic cord involvement. Histology was consistent with diffuse large B-cell lymphoma, germinal centre subtype. The patient was subsequently referred to haematology and following 3-cycles of chemotherapy demonstrated complete radiological response.As PTL carries a worse prognosis  than either germ cell tumours or NHL involving lymph-node groups or other extra-nodal sites, timely differentiation from other scrotal masses is required . TreatmeThe main differential diagnoses for PTL include germ cell tumours, acute and chronic epididymo-orchitis and leukaemia. Unlike lymphoma, germ cell tumours are frequently associated with raised serum Human chorionic gonadotropin (HCG) or alpha-fetoprotein (AFP) and metastasise in a predictable fashion cranially through successive retroperitoneal lymph-node groups . AlthougAt CT and ultrasound, sporadic nodal, and extra-nodal deposits around the body increase the probability of lymphoma over germ cell tumours or infection. As in the two cases presented here, cord-like soft tissue extending in a contiguous fashion from the testicular mass onto spermatic cord and gonadal vessels in the retroperitoneum is a very specific feature that has only ever been described in PTL.


Hepatitis C virus (HCV) infection unfavorably affects the survival of both renal patients undergoing hemodialysis and renal transplant recipients. In this subset of patients, the effectiveness and safety of different combinations of interferon-free direct-acting antiviral agents (DAAs) have been analyzed in several small studies. Despite fragmentary, the available data demonstrate that DAA treatment is safe and effective in eradicating HCV infection, with a sustained virologic response (SVR) rates nearly 95% and without an increased risk of allograft rejection. This review article analyzes the results of most published studies on this topic to favor more in-depth knowledge of the readers on the subject. We suggest, however, perseverating in this update as the optimal DAA regimen may not be proposed yet, because of the expected arrival of newer DAAs and of the lack of data from large multicenter randomized controlled trials. Hepatitis C virus (HCV) infection affects nearly 150 million people worldwide and more than 350,000 people die each year of HCV-related diseases . In the Interferon-based regimens had been the backbone of HCV treatment for kidney transplant recipients until 2014, a therapeutic approach limited by the relatively low efficacy in achieving viral eradication, poor tolerability , 52, andth week of treatment and throughout a subsequent follow-up of 12 weeks.The first-generation direct-acting antivirals (DAAs) protease inhibitors telaprevir and boceprevir present a similar limitation because their efficacy depends on the coadministration of interferon –63. InstA clear example of the efficacy of DAAs in eradicating HCV infection in patients with chronic kidney diseases (CKD) is offered by the C-SURFFER study, a multicenter, double-blind, randomized study where 224 patients with HCV GT1 infection and CKD stages 4 or 5 were randomly assigned to an “immediate treatment group” receiving grazoprevir and elbasvir (n=111) for 12 weeks or to a “deferred treatment” group treated with the same schedule (n=113) 16 weeks later. Most of patients were haemodialysis dependent; half of them had HCV genotype 1a infection, 80% were HCV treatment naïve, and 6% had liver cirrhosis. SVR rates in the “immediate treatment” and “deferred treatment” groups were 99% and 98, respectively .There are four classes of direct-acting antiviral drugs that combine the attack on more than one HCV life cycle goal: (1) NS3/4A protease inhibitors ; (2) the nucleoside and nucleotide NS5B polymerase inhibitors (sofosbuvir); (3) NS5A inhibitors ; and (4) nonnucleoside NS5B polymerase inhibitors (dasabuvir).General symptoms  associated with DAA treatment in kidney transplant recipients have been reported in nearly 40% of treated patients, whereas severe adverse events  are infrequent events (around 1% of treated patients) .Glomerular disease after DAAs has been also described. Lubetzky et al.  reportedIn this review article we analyze the data from several real-life studies on the feasibility, effectiveness, and tolerability of DAA therapies for kidney transplant recipients. The experience of most single studies is limited, prevalently by the low number of patients examined, but a comprehensive analysis of the available data can give useful indications to the readers –75.Episodes of reactivation of an indolent (open or occult) Hepatitis B virus (HBV) infection in patients with HBV/HCV-related chronic hepatitis during or after a successful DAA therapy have been documented . HBV reaIn HCV-related liver diseases, the combined use of two or more second-generation DAAs allows the eradication of HCV infection in nearly 95% of treated patients –83, a peThe knowledge on HCV eradication with DAA therapy in HCV-positive renal recipients or in those on a waiting list for renal transplantation is fragmentary, but the published data are highly promising. For HCV-positive patients in the wait list for kidney transplantation the first decision is whether to treat them before or after transplantation. For HCV patients with mild or moderate hepatic fibrosis who have a living kidney donor, treatment with DAA has been suggested prior to kidney transplantation , 67. ForAll this considered, it is commonly accepted that HCV-infected transplant candidates may receive a kidney from an HCV-positive donor. This eventuality had occurred in about 2500 donations in the USA between 2005 and 2014 ; otherwiAt present, no valid recommendation comes from prospective trials, but several studies, prevalently small and from single clinical centers, provide useful information on the effectiveness and tolerability of different DAAs combination regimens in HCV-positive transplant recipients.In a retrospective, multicentric study Fernández et al  observedLin et al.  treated Eisenberger et al.  assessedKamar et al.  treated Bhamidimarri et al.  describeSawinski et al.  used 4 dColombo et al.  carried Londono et al.  describeGallegos-Orozco et al.  describeDurand et al.  conducteGoldberg et al.  evaluateThe DAA regimens for HCV-positive renal transplant recipients should be adequate to HCV genotype, the entity of liver disease, the quality of posttransplant renal function, and the immunosuppressive drugs administered. A close monitoring of renal function during the DAA administration, with adjustment of DAA doses to the patients' eGFR, and a careful observation of a possible onset of drug to drag interactions, with adjustment of doses of immunosuppressive drugs, were recommended. For example, sofosbuvir-based DAA regimens may induce renal adverse events in patients with reduced renal function, particularly in those in an end-stage renal disease (ESRD) , 94, whiCalcineurin inhibitors (tacrolimus and ciclosporin) and the mechanized target of rapamycin inhibitors (everolimus and sirolimus), used as immunosuppressants in patients undergoing renal transplantation, are substrates of cytochrome P450 and therefore the possible interactions between these drugs and DAAs should always be taken into consideration , 96–98. The optimal timing of HCV therapy (pretransplant versus posttransplant) in HCV-positive transplant recipients is topic of debate. Several studies have shown that the wait times for kidney transplantation from HCV-positive donors are significantly shorter than those for kidneys from uninfected donors. These considerations support the use of all kidneys available for transplantation in HCV-positive patients, both HCV-negative and HCV-positive.The Kidney Disease Improving Global Outcomes guidelines currently recommend treating with DAAs the HCV RNA positive candidates for renal transplant while still in dialysis , since oBecause of the persistent shortage of kidneys that can be transplanted, it has been proposed to transplant HCV-positive kidneys into HCV-negative recipients and to initiate treatment with DAA immediately after transplantation, before the liver disease develops. However, although a few preliminary studies have provided encouraging results, the ethical implications of this procedure should be carefully evaluated, since approximately 5% of patients may not obtain SVR12 with DAA treatment; in some cases the donor HCV genotype may be difficult to treat one  and in very rare cases fibrosing cholestatic hepatitis may develop after kidney transplantation.A careful assessment of the stage of liver disease should be made including an accurate anamnesis, complete physical examination, serial determinations of serum liver function tests , upper abdominal ultrasound examination, and endoscopic examination of the upper intestinal tract in cirrhotic patients. Patients with advanced fibrosis should be regularly revaluated.The occurrence of reactivation of an indolent (open or occult) Hepatitis B virus (HBV) infection in HBV/HCV kidney‐transplant recipients  during oMost DAA therapeutic regimens are practically restricted to HCV genotypes 1 and 4, while HCV genotype 3 is quite frequent and equally dangerous. This need will be overcome by the newer pan-genotypic DAAs regimens (glecaprevir/pibrentasvir).The optimal regimens to be applied either before or after kidney transplantation may not be proposed yet, because of the expected arrival of other newer DAAs and of results from multicenter randomized controlled trials. Newer DAA regimens requiring a shorter duration of treatment may also accelerate the access to transplantation.The possibility to transplant HCV-positive kidneys into HCV-negative recipients has been recently analyzed by the American Society of Transplantation Consensus Conference on HCV-positive donors and kidney transplantation. It has been considered that the high level of safety and efficacy of DAAs in eradicating HCV infection provides the opportunity to explore their use in transplanting kidneys from HCV-viremic patients into non-HCV-viremic recipients, a practice that could save the life of numerous CKD patients with organ failure. Although more research is needed before making a final settlement on this topic, the consensus underlines the need to guarantee the access to DAA therapy to the HCV-negative recipients at the time they will receive a kidney from an HCV-positive donor.It has been also underlined that a living donor who has cleared HCV infection after DAA treatment does not transmit HCV infection to the kidney recipients –105.Treating HCV infection before kidney transplantation could be potentially advantageous, since eradicating HCV would lead to better allograft and patient survival.Ideally, patients with ESRD and HCV infection should be comanaged by surgeons, nephrologists, and hepatologists before and after transplantation.