Rose bengal-encapsulated chitosan nanoparticles for the photodynamic treatment of Trichophyton species.

Rose bengal (RB) solutions coupled with a green laser have proven to be efficient in clearing resilient nail infections caused by Trichophyton rubrum in a human pilot study and in extensive in vitro experiments. Nonetheless, the RB solution can become diluted or dispersed over the tissue and prevented from penetrating the nail plate to reach the subungual area where fungal infection proliferates. Nanoparticles carrying RB can mitigate the problem of dilution and are reported to effectively penetrate through the nail. For this reason, we have synthesized RB-encapsulated chitosan nanoparticles with a peak distribution size of ~200 nm and high reactive oxygen species (ROS) production. The RB-encapsulated chitosan nanoparticles aPDT were shown to kill more than 99% of T. rubrum, T. mentagrophytes, and T. interdigitale spores, which are the common clinically relevant pathogens in onychomycosis. These nanoparticles are not cytotoxic against human fibroblasts, which promotes their safe application in clinical translation.

On cooperative libertines and wicked puritans.

We agree with Fitouchi et al. that self-denial is sometimes moralized to signal capacity for cooperation, but propose that a person's cooperative character is more precisely judged by willingness to follow cultural, group, and interpersonal goals, for which many rules can serve as proxies, including rules about abstention. But asceticism is not a moral signal if its aims are destructive, while indulging impulses in a culturally approved way can also signal cooperation.

Relating pathogenic loss-of-function mutations in humans to their evolutionary fitness costs.

Causal loss-of-function (LOF) variants for Mendelian and severe complex diseases are enriched in 'mutation intolerant' genes. We show how such observations can be interpreted in light of a model of mutation-selection balance and use the model to relate the pathogenic consequences of LOF mutations at present to their evolutionary fitness effects. To this end, we first infer posterior distributions for the fitness costs of LOF mutations in 17,318 autosomal and 679 X-linked genes from exome sequences in 56,855 individuals. Estimated fitness costs for the loss of a gene copy are typically above 1%; they tend to be largest for X-linked genes, whether or not they have a Y homolog, followed by autosomal genes and genes in the pseudoautosomal region. We compare inferred fitness effects for all possible de novo LOF mutations to those of de novo mutations identified in individuals diagnosed with one of six severe, complex diseases or developmental disorders. Probands carry an excess of mutations with estimated fitness effects above 10%; as we show by simulation, when sampled in the population, such highly deleterious mutations are typically only a couple of generations old. Moreover, the proportion of highly deleterious mutations carried by probands reflects the typical age of onset of the disease. The study design also has a discernible influence: a greater proportion of highly deleterious mutations is detected in pedigree than case-control studies, and for autism, in simplex than multiplex families and in female versus male probands. Thus, anchoring observations in human genetics to a population genetic model allows us to learn about the fitness effects of mutations identified by different mapping strategies and for different traits.

Calcium-Mediated Calpain Activation and Microtubule Dissociation in Cell Model of Hereditary Sensory Neuropathy Type-1 Expressing V144D SPTLC1 Mutation.

Hereditary sensory neuropathy type 1A (HSN1A) is an autosomal, dominantly inherited peripheral neuropathy caused by mutations in serine palmitoyl transferase long chain 1 (SPTLC1), involved in the de novo synthesis of sphingolipids. We have previously reported calcium imbalance, as well as mitochondrial and ER stress in both HSN1 patient lymphoblasts and a transiently transfected cell model. In this study, we investigated the role of the Ca2+-activated protease calpain in destabilizing the cell cytoskeleton, by examining calpain activity in SH-SY5Y cells overexpressing the V144D mutant and changes in microtubule-associated proteins (MAP). Intramitochondrial Ca2+ was found to be significantly depleted and cytoplasmic Ca2+ increased in the V144D mutant. Subsequently, calpain and proteasome activity were increased and calpain substrates, microtubule associated proteins MAP2, and tau were significantly reduced in the microtubule fraction of the mutant. Significant changes were also found in motor proteins dynein and KIF2A detected in the microtubule fraction of cells overexpressing the V144D mutation. There was also a reduction in anterograde and retrograde mitochondrial transport velocities in the V144D mutant. These findings strongly implicate cytoskeletal aberration caused by Ca2+ dysregulation and subsequent loss of microtubule transport functions as the cause of axonal dying back that is characteristic of HSN1.

Altering the Binding Properties of PRDM9 Partially Restores Fertility across the Species Boundary.

Sterility or subfertility of male hybrid offspring is commonly observed. This phenomenon contributes to reproductive barriers between the parental populations, an early step in the process of speciation. One frequent cause of such infertility is a failure of proper chromosome pairing during male meiosis. In subspecies of the house mouse, the likelihood of successful chromosome synapsis is improved by the binding of the histone methyltransferase PRDM9 to both chromosome homologs at matching positions. Using genetic manipulation, we altered PRDM9 binding to occur more often at matched sites, and find that chromosome pairing defects can be rescued, not only in an intersubspecific cross, but also between distinct species. Using different engineered variants, we demonstrate a quantitative link between the degree of matched homolog binding, chromosome synapsis, and rescue of fertility in hybrids between Mus musculus and Mus spretus. The resulting partial restoration of fertility reveals additional mechanisms at play that act to lock-in the reproductive isolation between these two species.

Inferring Population Histories for Ancient Genomes Using Genome-Wide Genealogies.

Ancient genomes anchor genealogies in directly observed historical genetic variation and contextualize ancestral lineages with archaeological insights into their geography and cultural associations. However, the majority of ancient genomes are of lower coverage and cannot be directly built into genealogies. Here, we present a fast and scalable method, Colate, the first approach for inferring ancestral relationships through time between low-coverage genomes without requiring phasing or imputation. Our approach leverages sharing patterns of mutations dated using a genealogy to infer coalescence rates. For deeply sequenced ancient genomes, we additionally introduce an extension of the Relate algorithm for joint inference of genealogies incorporating such genomes. Application to 278 present-day and 430 ancient DNA samples of >0.5x mean coverage allows us to identify dynamic population structure and directional gene flow between early farmer and European hunter-gatherer groups. We further show that the previously reported, but still unexplained, increase in the TCC/TTC mutation rate, which is strongest in West Eurasia today, was already present at similar strength and widespread in the Late Glacial Period ~10k-15k years ago, but is not observed in samples >30k years old. It is strongest in Neolithic farmers, and highly correlated with recent coalescence rates between other genomes and a 10,000-year-old Anatolian hunter-gatherer. This suggests gene-flow among ancient peoples postdating the last glacial maximum as widespread and localizes the driver of this mutational signal in both time and geography in that region. Our approach should be widely applicable in future for addressing other evolutionary questions, and in other species.

Rapid genotype imputation from sequence with reference panels.

Inexpensive genotyping methods are essential to modern genomics. Here we present QUILT, which performs diploid genotype imputation using low-coverage whole-genome sequence data. QUILT employs Gibbs sampling to partition reads into maternal and paternal sets, facilitating rapid haploid imputation using large reference panels. We show this partitioning to be accurate over many megabases, enabling highly accurate imputation close to theoretical limits and outperforming existing methods. Moreover, QUILT can impute accurately using diverse technologies, including long reads from Oxford Nanopore Technologies, and a new form of low-cost barcoded Illumina sequencing called haplotagging, with the latter showing improved accuracy at low coverages. Relative to DNA genotyping microarrays, QUILT offers improved accuracy at reduced cost, particularly for diverse populations that are traditionally underserved in modern genomic analyses, with accuracy nearly doubling at rare SNPs. Finally, QUILT can accurately impute (four-digit) human leukocyte antigen types, the first such method from low-coverage sequence data.

Enhanced Recovery Pathways for Flap-Based Reconstruction: Systematic Review and Meta-Analysis.

BACKGROUND: Enhanced Recovery After Surgery (ERAS) pathways are known to improve patient outcomes after surgery. In recent years, there have been growing interest in ERAS for reconstructive surgery. OBJECTIVES: To systematically review and summarise literature on the key components and outcomes of ERAS pathways for autologous flap-based reconstruction. DATA SOURCES: Cochrane Central Register of Controlled Trials, MEDLINE, EMBASE, Current Controlled Trials, World Health Organization International Clinical Trials Registry Platform and reference lists of relevant studies. INCLUSION CRITERIA: All primary studies of ERAS pathways for free and pedicled flap-based reconstructions reported in the English language. OUTCOME MEASURES: The primary outcome measure was length of stay. Secondary outcomes were complication rates including total flap loss, partial flap loss, unplanned reoperation within 30 days, readmission to hospital within 30 days, surgical site infections and medical complications. RESULTS: Sixteen studies were included. Eleven studies describe ERAS pathways for autologous breast reconstructions and five for autologous head and neck reconstructions. Length of stay was lower in ERAS groups compared to control groups (mean reduction, 1.57 days; 95% CI, - 2.15 to - 0.99). Total flap loss, partial flap loss, unplanned reoperations, readmissions, surgical site infections and medical complication rates were similar between both groups. Compliance rates were poorly reported. CONCLUSION: ERAS pathways for flap-based reconstruction reduce length of stay without increasing complication rates. ERAS pathways should be adapted to each institution according to their needs, resources and caseload. There is potential for the development of ERAS pathways for chest wall, perineum and lower limb reconstruction. LEVEL OF EVIDENCE III: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Microvascular anastomotic arterial coupling: A systematic review.

INTRODUCTION: There are several reasons microsurgeons may not use a coupler device in arterial anastomosis: may be thick-walled, non-pliable due to atherosclerotic calcification or present vessel geometrical discrepancies. This review summarises the current applications, efficacy and troubleshooting of microvascular coupler devices in arterial end-to-end anastomosis. METHODS: A systematic review of the literature was performed in November 2020 across 4 electronic databases and in accordance with the PRISMA guidelines. All studies comprised the data synthesis that reported the use of a microvascular coupler device for arterial end-to-end anastomosis. Data were extracted and collected in three groups of standardised variables: study, anastomosis-related and technical characteristics. RESULTS: Out of the 7,690 articles identified, 20 were included in the final data synthesis. Included studies involved a total of 1639 patients, who underwent 670 arterial and 1,124 venous anastomoses. Out of all arterial anastomoses, 351 were performed in free tissue transfers in head and neck, 117 in breast, 4 in upper extremity and 5 in lower extremity reconstruction, whereas the remaining were not specified. The total arterial coupler anastomosis success rate reported was 92.1% (617/670). Fifty-three (8%) arterial anastomoses were reported to result in either troubleshooting events or intra- or post-operative failures, most being reported in extremity reconstructions. CONCLUSIONS: Arterial coupling is not widespread with predominant use in head and neck and chest reconstructions, and total reported efficacy of 92.1%. Microsurgeons are reluctant to routinely use current widespread coupler devices as a result of inherent arterial characteristics. This study delivered collective recommendations, 'do's and don'ts' of microvascular arterial coupling.

Recycling of flap pedicle in complex lower extremity reconstruction: A proof of free muscle flap neovascularization.

This article portrays the authors' clinical experience of a complex case of lower extremity reconstruction using a recycled pedicle from 10 years old free latissimus dorsi musculocutaneous flap to supply a new free anterolateral thigh flap for proximal tibia wound defect reconstruction. It provides clinical evidence that muscle neovascularization occurs and supports the dogma peripheral tissue neovascularization. This case stipulates that recycling of pedicle is feasible, when used with appropriate strategy and safety and also provides evidence for the long-term survival of greater saphenous vein grafts in lower extremity reconstruction.

The "Crater" Arteriotomy: A Technique Aiding Precise Intimal Apposition in End-to-side Microvascular Anastomosis.

End-to-side arterial anastomoses require a high level of technical competency. The main challenge to a successfully patent anastomosis is intimal interposition during the standardized microvascular suturing. Technical errors during arteriotomy pose a significant challenge for the microsurgical technique, making the end-to-side anastomosis prone to failure. We describe a basic yet fundamental method of performing an arteriotomy, the "crater" technique, which facilitates good visualization of all vessel layers before placement of microsurgical sutures. Using curved microsurgical scissors, the adventitia layer is dissected off the outer surface of the side vessel, a V-shaped cut is then made obliquely at a 30-45 degrees angle to the longitudinal axis of the vessel, and a full thickness oblique cut is made along an elliptical circumference, as the curved scissors enable the creation of a slope-like crater. This concept ensures the intimal layer is adequately exposed through the complete circumference of the arteriotomy rim, while enabling a variable increase in the arterial wall hypotenuse-width circumference. When performed in a standardized manner, the crater arteriotomy can minimize the risk of endothelial misalignment and further technical errors during suturing, thus minimizing the risk of anastomotic failure.

ZCWPW1 is recruited to recombination hotspots by PRDM9 and is essential for meiotic double strand break repair.

During meiosis, homologous chromosomes pair and recombine, enabling balanced segregation and generating genetic diversity. In many vertebrates, double-strand breaks (DSBs) initiate recombination within hotspots where PRDM9 binds, and deposits H3K4me3 and H3K36me3. However, no protein(s) recognising this unique combination of histone marks have been identified. We identified Zcwpw1, containing H3K4me3 and H3K36me3 recognition domains, as having highly correlated expression with Prdm9. Here, we show that ZCWPW1 has co-evolved with PRDM9 and, in human cells, is strongly and specifically recruited to PRDM9 binding sites, with higher affinity than sites possessing H3K4me3 alone. Surprisingly, ZCWPW1 also recognises CpG dinucleotides. Male Zcwpw1 knockout mice show completely normal DSB positioning, but persistent DMC1 foci, severe DSB repair and synapsis defects, and downstream sterility. Our findings suggest ZCWPW1 recognition of PRDM9-bound sites at DSB hotspots is critical for synapsis, and hence fertility.

Sexual reproduction ­ that is, the combination of sex cells from two different individuals to produce an embryo ­ is one of the many mechanisms that have evolved to maintain genetic diversity. Most human cells contain 23 pairs of chromosomes, with each chromosome in a pair carrying either a paternal or maternal copy of the same gene. To form an embryo with the right number of chromosomes, each sex cell (the egg or sperm cell) must only contain one chromosome from each pair. Sex cells are produced from parent cells containing two sets of paternal and maternal chromosomes: these cells then divide twice to form four sex cells which contain only one chromosome from each pair. Before the parent cell divides, a process known as 'recombination' takes place, which allows chromosomes in a pair to exchange bits of genetic information. This reshuffling ensures that each chromosome in a sex cell is unique. A protein called PRDM9 helps control which sections of genetic information are recombined by modifying proteins attached to the chromosomes, marking them as locations for exchange. The DNA at each of these sites is then broken and repaired using the genetic sequence of the chromosome it is paired with as a template, thus causing the two chromosomes to swap genes. In 2019, a group of researchers found a set of genes in the testis of mice that are expressed at the same time as the gene for PRDM9. This suggested that another protein called ZCWPW1 is likely involved in recombination, but the precise role of this protein was unclear. To answer this question, Wells, Bitoun et al. ­ including many of the researchers involved in the 2019 study ­ examined human cells grown in the laboratory to determine where ZCWPW1 binds to in the chromosome. This revealed that ZCWPW1 can be found at the same sites as PRDM9, which is responsible for bringing it there. Furthermore, cells from male mice lacking the gene for ZCWPW1 cannot complete the exchange of genetic information between chromosomes, meaning that the mice are infertile. As such, ZCWPW1 seems to connect location selection by PRDM9 to the DNA repair mechanisms needed for gene exchange between chromosomes. Infertility is a significant issue for humans affecting as many as one in every six couples. Fertility is complex and many of the biological mechanisms involved are not fully understood. This work suggests that both PRDM9 and ZCWPW1 are key to the production of sex cells and may be worth investigating as factors that affect fertility in humans.

The Exoscope versus operating microscope in microvascular surgery: A simulation non-inferiority trial.

BACKGROUND: The Exoscope is a novel high-definition digital camera system. There is limited evidence signifying the use of exoscopic devices in microsurgery. This trial objectively assesses the effects of the use of the Exoscope as an alternative to the standard operating microscope (OM) on the performance of experts in a simulated microvascular anastomosis. METHODS: Modus V Exoscope and OM were used by expert microsurgeons to perform standardized tasks. Hand-motion analyzer measured the total pathlength (TP), total movements (TM), total time (TT), and quality of end-product anastomosis. A clinical margin of TT was performed to prove non-inferiority. An expert performed consecutive microvascular anastomoses to provide the exoscopic learning curve until reached plateau in TT. RESULTS: Ten micro sutures and 10 anastomoses were performed. Analysis demonstrated statistically significant differences in performing micro sutures for TP, TM, and TT. There was statistical significance in TM and TT, however, marginal non-significant difference in TP regarding microvascular anastomoses performance. The intimal suture line analysis demonstrated no statistically significant differences. Non-inferiority results based on clinical inferiority margin (Δ) of TT=10 minutes demonstrated an absolute difference of 0.07 minutes between OM and Exoscope cohorts. A 51%, 58%, and 46% improvement or reduction was achieved in TT, TM, TP, respectively, during the exoscopic microvascular anastomosis learning curve. CONCLUSIONS: This study demonstrated that experts' Exoscope anastomoses appear non-inferior to the OM anastomoses. Exoscopic microvascular anastomosis was more time consuming but end-product (patency) in not clinically inferior. Experts' "warm-up" learning curve is steep but swift and may prove to reach clinical equality.

The end game - A quantitative assessment tool for anastomosis in simulated microsurgery.

With the move towards simulation based microsurgical training and emphasis on the declining usage of animal models, there is a need for an objective method to evaluate microvascular anastomosis in a non-living, simulated microsurgical training environment. Our aim was to create a validated assessment tool to evaluate the intimal surface of the end product to measure skills acquisition. The intimal surface of 200 anastomoses from 23 candidates and 2 experts were assessed using ImageJ to measure 4 parameters: 1) distance between the distal insertion points, 2) distance between the proximal insertion points, 3) length of sutures placed, 4) number of axes. Using these parameters, a 9-component scoring system was produced based on the hypothesis of the ideal anastomosis having equidistance between the above parameters. The scoring system was devised based on population performance to give a maximum score of 100. The EPIA tool demonstrated its ability to differentiate between seniority from undergraduate to expert. Furthermore, predictive validity was shown by demonstrating skill acquisition between day 3 and 5 of the microsurgery course. The EPIA tool is a valid and feasible method to assess and provide feedback regarding the end product as an adjunct to current scoring systems in simulated microsurgery.

International microsurgery simulation society (IMSS) consensus statement on the minimum standards for a basic microsurgery course, requirements for a microsurgical anastomosis global rating scale and minimum thresholds for training.

INTRODUCTION: Microsurgery is a surgical technique that uses optical magnification as well as specific instruments to address necessary reconstructive procedures in different medical specialties. The apprenticeship of this technique requires overcoming a steep learning curve. There is a need for standardization of the training criteria in microsurgery. The International Microsurgery Simulation Society (IMSS) was born in 2011, since then its main objective has been to connect the main international specialists and educators of this sub-specialty to share and discuss the ethical and scientific basis of preclinical microsurgery teaching. METHODS: In order to achieve a consensus on the minimum standards for the organization of basic microsurgery training courses, the requirements for a microsurgical anastomosis global rating scale and minimum thresholds for training, a total of nineteen independent global experts participated in a formal consultative consensus development program. The agreement criteria for each statement was established when consensus of 65-100% was reached. RESULTS: There have been established six recommendations concerning minimum standards for a basic microsurgery course, one recommendation in relation to minimum thresholds for training and four recommendations regarding the global rating scale as gold standard for a microsurgical anastomosis assessment. The eleven defined recommendations reached the agreement threshold of 65-100%. CONCLUSIONS: The development of this consensus sets the minimum recommended requirements for conducting basic microsurgery training courses, as well as suggestions for objective assessment of the learning curve and skills of trainees.

The "Needle-splint" Technique: A Method of Accurate Apposition and Eversion during Microvascular Anastomosis.

We present the "needle-splint" technique, a microsurgical suturing technique that enhances micro-suturing technique, while ensures finer apposition and vessels walls eversion during the placement of sutures in microvascular anastomosis. This report demonstrates the usefulness of this technique in simple interrupted, continuous-interrupted, or multi-loops microsurgical suturing. It further allows direct visualization of the intra-wall-edges space and intimal sutures surface and could be utilized as a safety stabilizer to errors as it allows optimal vessels alignment while the needle curvature is acting as a "pusher" to separate the posterior wall during knot tying.

Porous chitosan adhesives with L-DOPA for enhanced photochemical tissue bonding.

L-3,4-dihydroxyphenylalanine (L-DOPA) is a naturally occurring catechol that is known to increase the adhesive strength of various materials used for tissue repair. With the aim of fortifying a porous and erodible chitosan-based adhesive film, L-DOPA was incorporated in its fabrication for stronger photochemical tissue bonding (PTB), a repair technique that uses light and a photosensitiser to promote tissue adhesion. The results showed that L-DOPA did indeed increase the tissue bonding strength of the films when photoactivated by a green LED, with a maximum strength recorded of approximately 30 kPa, 1.4 times higher than in its absence. The addition of L-DOPA also did not appreciably change the swelling, mechanical and erodible properties of the film. This study showed that strong, porous and erodible adhesive films for PTB made from biocompatible materials can be obtained through a simple inclusion of a natural additive such as L-DOPA, which was simply mixed with chitosan without any chemical modifications. In vitro studies using human fibroblasts showed no negative effect on cell proliferation indicating that these films are biocompatible. The films are convenient for various surgical applications as they can provide strong tissue support and a microporous environment for cellular infusion without the use of sutures. STATEMENT OF SIGNIFICANCE: Tissue adhesives are not as strong as sutures on wounds under stress. Our group has previously demonstrated that strong sutureless tissue repair can be realised with chitosan-based adhesive films that photochemically bond to tissue when irradiated with green light. The advantage of this technique is that films are easier to handle than glues and sutures, and their crosslinking reactions can be controlled with light. However, these films are not optimal for high-tension tissue regenerative applications because of their non-porous structure, which cannot facilitate cell and nutrient exchange at the wound site. The present study resolves this issue, as we obtained a strong and porous photoactivated chitosan-based adhesive film, by simply using freeze drying and adding L-DOPA.

Fractional radiofrequency in the treatment of skin aging: an evidence-based treatment protocol.

Fractional radiofrequency (FRF) has recently emerged for the treatment of scars, cellulite and skin rejuvenation. The aim of this paper was to investigate the evidence behind its use in skin aging and evaluate efficacy, safety, and standardization of protocols. The literature was systematically searched and finally 25 full-text articles were included. Two were randomized controlled trials, 3 were comparative studies, and 20 were case series. Most studies were underpowered with low methodological quality. The participants had skin phototype I-VI with variable baseline severity of signs. Fractional radiofrequency using microneedles or electrode pins was performed on the face, neck, and décolletage. There was heterogeneity in outcomes measurement, but the efficacy of FRF was confirmed in all relevant studies. Mainly, the improvement of rhytides and skin tightening were reported. Mild to moderate pain, transient erythema and edema were the commonest adverse events. Hyperpigmentation was also noted in some cases. There was no consistency in the protocols used and in the description of procedures. A clinical impact score was created to assess the studies and to aid the generation of an evidence-based protocol for minimally invasive radiofrequency procedures. However, there is a need for large scale, well-designed trials to better investigate the efficacy and safety of FRF and to produce clear guidelines.

Suppression of the Peripheral Immune System Limits the Central Immune Response Following Cuprizone-Feeding: Relevance to Modelling Multiple Sclerosis.

Cuprizone (CPZ) preferentially affects oligodendrocytes (OLG), resulting in demyelination. To investigate whether central oligodendrocytosis and gliosis triggered an adaptive immune response, the impact of combining a standard (0.2%) or low (0.1%) dose of ingested CPZ with disruption of the blood brain barrier (BBB), using pertussis toxin (PT), was assessed in mice. 0.2% CPZ(±PT) for 5 weeks produced oligodendrocytosis, demyelination and gliosis plus marked splenic atrophy (37%) and reduced levels of CD4 (44%) and CD8 (61%). Conversely, 0.1% CPZ(±PT) produced a similar oligodendrocytosis, demyelination and gliosis but a smaller reduction in splenic CD4 (11%) and CD8 (14%) levels and no splenic atrophy. Long-term feeding of 0.1% CPZ(±PT) for 12 weeks produced similar reductions in CD4 (27%) and CD8 (43%), as well as splenic atrophy (33%), as seen with 0.2% CPZ(±PT) for 5 weeks. Collectively, these results suggest that 0.1% CPZ for 5 weeks may be a more promising model to study the 'inside-out' theory of Multiple Sclerosis (MS). However, neither CD4 nor CD8 were detected in the brain in CPZ±PT groups, indicating that CPZ-mediated suppression of peripheral immune organs is a major impediment to studying the 'inside-out' role of the adaptive immune system in this model over long time periods. Notably, CPZ(±PT)-feeding induced changes in the brain proteome related to the suppression of immune function, cellular metabolism, synaptic function and cellular structure/organization, indicating that demyelinating conditions, such as MS, can be initiated in the absence of adaptive immune system involvement.