Heavy metal (HM) contamination of the environment is a major problem worldwide. The rate of global deposition of HMs in soil has dramatically increased over the past two centuries and there of facilitated their rapid accumulation also in living systems. Although the effects of HMs on plants, animals and humans have been extensively studied, yet little is known about their effects on the (patho)biology of the microorganisms belonging to a unique group of filamentous eukaryotic pathogens, i.e., fungi and oomycetes. Much of the literature concerning mainly model species has revealed that HM stress affects their hyphal growth, morphology, and sporulation. Toxicity at cellular level leads to disturbance of redox homeostasis manifested by the formation of nitro-oxidative intermediates and to the induction of antioxidant machinery. Despite such adverse effects, published data is indicative of the fact that fungal and oomycete pathogens have a relatively high tolerance to HMs in comparison to other groups of microbes such as bacteria. Likely, these pathogens may harbor a network of detoxification mechanisms that ensure their survival in a highly HM-polluted (micro)habitat. Such a network may include extracellular HMs immobilization, biosorption to cell wall, and/or their intracellular sequestration to proteins or other ligands. HMs may also induce a hormesis-like phenomenon allowing the pathogens to maintain or even increase fitness against chemical challenges. Different scenarios linking HMs stress and modification of the microorganisms pathogenicity are disscused in this review. Fungi and oomycetes represent eukaryotic microbes that are characterized by filamentous vegetative hyphae networks, form spores for asexual and sexual reproduction and share similar processes of infection and nutrition acquisition , iron (Fe), zinc (Zn)] and (2) non-essential elements of an unknown biological role but adversely affect the organism . HMs occur naturally in the environment, the natural sources include weathering of metal-containing rocks and volcanic eruptions. The increase in agricultural and industrial activities has dramatically accelerated the environmental pollution due to HMs while the anthropogenic origins have contributed to the dispersal of HMs in soil, water and air organisms. They include two groups: (1) essential elements that in small amounts are crucial for the physiological functions of organisms but toxic when present in excess  at concentrations that ranged from 10–4 to 10–5 M observed the inhibition of germ tube growth and spore germination. The most severe inhibitory effects were found due to Hg, Cd, or Cu (Bhajbhuje −1 Ag and Cu) independently tested against plant pathogenic fungi B. cinerea and A. alternata led to inhibition of their hyphal growth  caused abnormal spiral growth of the hyphae  caused encystment of 90% zoospores  that result in oxidative and/or nitrosative stress and disturbance of the cell redox balance  and Cd (50 µM) independently applied caused ROS formation and time-dependent changes in the activity of CAT, POX, lignin peroxidase (LiP), and manganese peroxidase (MnP). The intracellular enzymes such as CAT and POX showed a similar trend in the activity, however, HM-mediated induction of CAT and POX was lower than that in LiP and MnP  and Zn (0.1–20 mg/L) provoked elevation of CAT and SOD activities at the selected HM dose (1 mg/L), while concentrations above this threshold gradually diminished the enzyme activity  stimulate mycelial growth of phytopathogenic fungus Endothia parasitica (Englander and Corden A. flavus at relatively low concentration (5 mg L−1) were found to stimulate not only the mycelial growth of the pathogen but also aflatoxin biosynthesis. Moreover, the accumulation of total RNA was enhanced by Cu, Zn and Fe; however, the combination of all three HMs or their duplexes (Zn + Cu and Zn + Fe) were much more effective in total RNA accumulation as compared to the control , although the latter is not an HM . Sporangia in both S. delica and D. carpophorus formed in the presence of Zn were elongated compared to the control , the oxalic acid is more predominant in fungi  at 400 mg/l increased oxalic acid content by 3.5-fold  transporter family is involved in intracellular transfer of HMs in fungi  and metallothionein’s (MTs). Being thiol compounds they are the prime agents for cellular HMs tolerance and involvement of MTs in fungal HM detoxification. Metallothioneins are low molecular weight cysteine rich proteins that coordinately bind divalent or monovalent metal ions and control the homeostasis of essential metals such as Zn and Cu, or lead to sequestration of the non-essential toxic metals such as Cd  was found in the genome of Lindgomycetaceae P87 and Curvularia geniculata P1 were found to reduce mercury ion Hg (II) and the reaction led to the formation of volatile forms of Hg enabling its evaporation  adsorption of Cr (VI) by carboxyl, hydroxide, amine, amide, cyano and phosphate groups of cell wall, and (ii) the Cr (VI) reduction to Cr (III), which is sparingly soluble in water form, and therefore not toxic to the cell  expression revealed an elevated PiTef1 gene expression in planta in each tested potato cultivar; however, the level of PiTef1 expression depended on the degree of basal resistance of the potato cultivars used in this study  showed limited pathogenicity in the presence of Cd or Pb ions, whereas independently applied Cu, Zn, or Ni ions each had a weak or no effect on the fungal pathogenicity . Cd also limited pathogenicity of the entomopathogen fungus Isaria javanica. Under normal conditions, I. javanica caused mortality of two aphid species while after Cd exposure the pathogen caused 30–60% mortality of aphids , a metal transporter with potential to eliminate superoxide radicals, can be involved in metal metabolism and virulence. Also, deficiency of PcaA leads to decreased Cd tolerance and attenuates the virulence of A. fumigatus in the Galleria mellonella infection model, resulting in a decreased tolerance to oxidative stress  proteins during pulmonary infection  encoding MT-like protein resulted in diminished pathogenicity of hemibiotroph Magnaporthe grisea in rice that had a defect in the pre-penetration phase  from aqueous solution  to less toxic or nontoxic Cr (III)  (IV), uranium (U) (IV), and plutonium (Pu) (IV) but also by facilitating the biodegradation of petroleum hydrocarbons . Utilization of a heterogeneous group of microorganisms can be particularly helpful in remediating co-contaminated environments that are a serious global problem since HMs are also found in petroleum-contaminated soils. Thuse.g. Plasmopara viticola, P. infestans, as well as fungi such as A. solani and Elsinoë ampelina  Do HMs promote or limit pathogenicity and/or virulence and (2) what is the nature of the observed phenomenon? (3) What is the transcriptional response to the hormetic action of HMs? (4) Considering the huge biotechnological potential of fungal microorganisms—should research focus on those pathogenic fungi that have the potential for large scale bioremediation processes? There is a need to characterize the chemistry and environmental fates of the metabolites produced during fungal bioremediation. Finally, HMs are transferred from contaminated soil or water to animal and human organisms via the food chain, thus affecting their natural microbiota. A similar effect may be caused by uncontrolled dietary supplementation with commercial products containing essential elements such as Zn, Fe or Mn. (5) Future research should therefore focus on the identification of the effect of HMs against human and animal microbiota. The growing interest in such issues should help in broadening our knowledge on HM contamination of the microbial environment.


Risk factors of late-onset Alzheimer's disease (AD) such as aging, type 2 diabetes, obesity, heart failure, and traumatic brain injury can facilitate the appearance of cognitive decline and dementia by triggering cerebrovascular pathology and neuroinflammation.White matter (WM) microstructure and function are especially vulnerable to these conditions. Microstructural WM changes, assessed with diffusion weighted magnetic resonance imaging, can already be detected at preclinical stages of AD, and in the presence of the aforementioned risk factors. Particularly, the limbic system and cortico-cortical association WM tracts, which myelinate late during brain development, degenerate at the earliest stages. The fornix, a C-shaped WM tract that originates from the hippocampus, is one of the limbic tracts that shows early microstructural changes. Fornix integrity is necessary for ensuring an intact executive function and memory performance. Thus, a better understanding of the mechanisms that cause fornix degeneration is critical in the development of therapeutic strategies aiming to prevent cognitive decline in populations at risk. In this literature review, i) we deepen the idea that partial loss of forniceal integrity is an early event in AD, ii) we describe the role that common risk factors of AD can play in the degeneration of the fornix, and iii) we discuss some potential cellular and physiological mechanisms of WM degeneration in the scenario of cerebrovascular disease and inflammation. PreseWhite matter (WM) is known to be especially susceptible to vascular pathology, supposedly, due to a lower microvascular density and reduced blood supply compared to the gray matter The fornix is the main inflow/outflow information pathway of the hippocampus, making it a key structure in learning and memory processes. Reduced forniceal integrity already appears at preclinical phases of AD  plaques and neurofibrillary tangles (composed of hyperphosphorylated tau) as primary histological hallmarks. Since the early 1990s, the amyloid hypothesis has motivated the vast majority of clinical trials in AD Recently, the vascular component of AD has regained prominence. Epidemiological studies have associated some CVRFs  with a higher incidence of AD and, presumably, clinical strategies focused on CVRF prevention could reduce the predicted prevalence of AD in the long term ,35.A compromised cerebrovascular function is now recognized as a critical event in the development of late-onset AD, with up to 80% of AD patients showing vascular pathology . MoreoveIn addition, neuroimaging studies of diffusion tensor imaging (DTI) have pointed to disrupted functional networks, due to microstructural WM changes, as a possible cause of the earliest cognitive impairments in AD patients.In the WM, DTI studies rely on the highly ordered (anisotropic) water diffusion that results from the unique geometric organization of axonal fiber bundles. Changes in DTI parameters , radial diffusivity (RD), mean diffusivity (MD) and fractional anisotropy (FA)) refer to the altered anisotropy and/or diffusivity of water molecules caused by changes in the WM microstructure ,46. MD aInterestingly, AD patients with a strong vascular component show more severe microstructural WM changes than those with a subtle vascular component The disconnection hypothesis has added one more level of complexity to the understanding of AD etiology but, on the other hand, comprehending the physiological mechanisms that trigger WM changes and the link with vascular pathology may shed light on the relationship between the cerebrovascular dysfunction seen in many risk factors of AD and the neurodegeneration finally leading to AD.A better understanding of these mechanisms is also essential for novel therapies aiming to rescue dysfunctional brain circuits to succeed. Namely, deep brain stimulation of the fornix (DBS-f). DBS is a neurosurgical procedure able to modulate the activity of brain circuits through electrical stimulation delivered by electrodes that are implanted in target areas in the brain 3The fornix is an arch-shaped WM structure located under the corpus callosum and above the thalamus . It is cThe interruption of this circuitry as a consequence of fornix degeneration in aging or pathological conditions is believed to impair executive function and episodic memory consolidation The specific mechanism by which fornix degeneration impairs cognition and memory in humans are poorly understood, especially because, in most cases, lesions of the fornix are accompanied with lesions in other brain regions that could also be involved in learning and memory . But experimental animal models suggest dysregulation of the cholinergic and GABAergic signaling as possible factors The role of the fornix in memory recall has also been studied in healthy volunteers by using DTI 4WM degeneration in AD has been explained by two different mechanisms: Wallerian degeneration and the retrogenesis model ,83,84. TOn the contrary, the retrogenesis model assumes primary WM changes through myelin breakdown and axonal damage that develops in a reverse pattern of myelinogenesis during brain development ,83,84. TThe fact that fornix degeneration appears at preclinical phases of AD disease, and that it has a strategic location within the memory circuitry, makes it a good candidate for an early AD biomarker Whether microstructural WM changes in the fornix are the cause or consequence of the neurodegeneration occurring in the hippocampus, the main structural biomarker of neurodegeneration proposed by the National Institute on Aging-Alzheimer's Association The association between WM changes in the fornix and the main hallmark of AD (Aβ deposition in the brain) has been explored by some authors. Reduced FA in the fornix was correlated with Aβ deposition when assessed with positron emission tomography in both cognitively normal and MCI subjects ,100. AlsAPOE ε4 allele carriers, the strongest genetic factor known to increase the risk of late-onset AD ,105, havAll these studies support that WM changes in the fornix are present at a very early stage of the disease, and often precede the structural alterations occurring in the hippocampus, but it remains unclear what causes forniceal degeneration at such early stages. Chronic hypoperfusion, ischemic processes, neuroinflammation, demyelination and axonal loss are some putative factors proposed by DTI studies that can alter the microstructure of the WM tracts in AD ,107,108.5Brain network dynamics are modulated by daily life factors such as education, diet, and physical exercise 5.1Aging is the primary risk factor of neurodegeneration and dementia. Most DTI-based studies agree on WM microstructure being altered in elderly people ,22. The Impaired cerebrovascular hemodynamics and inflammation are some of the age-related factors that can contribute to WM damage. The increased pulse pressure waves penetrating the cerebral microcirculation as a result of reduced arterial myogenic responses, may contribute to WM damage, and it has been reported to precipitate cognitive deficits in the elderly 5.2CVRFs are linked to the appearance of vascular dementia and AD ,121. EpiPatients with type 2 diabetes, have a greater risk of suffering from vascular cognitive impairment and dementia and late-onset AD in the elderly f) from qMT, Metzler-Baddeley et al., Moreover, either in obese  or non-obese elder people, body mass index negatively correlated with WM integrity, namely in the fornix and posterior regions of the corpus callosum 5.3Populations with heart failure have lower cardiac output, and the reduced blood flow pumped by the heart seems to have a detrimental impact on cerebral circulation Kumar et al., found that the volumes of the mammillary bodies and the fornix were significantly reduced in patients with heart failure manifesting spatial and working memory deficits 5.4The most relevant consequences of TBI are diffuse axonal injury caused by mechanical forces (shear stress) and cerebrovascular dysfunction  ,138. NamAdult TBI patients (∼40 years old), showed persistent microglia activation ([11C](R)PK11195 (PK) binding in positron emission tomography imaging) in the internal capsule, putamen and, especially, in WM surrounding thalamic nuclei (fornix). However, only microglia activation in the latter correlated with lower executive function performance Intriguingly, in adolescent mild TBI patients (∼15 years old), Yallampalli et al., found an unexpected increased FA in acute phases, compared to controls, which they attributed to cytotoxicity and inflammation preceding myelin degeneration at more chronic phases These studies strongly support that cognitive deficits in TBI individuals are related to network dysfunction and disconnection and could facilitate the appearance of AD later on ,146.6Among the molecular and physiological mechanisms proposed as initiators of retrogenesis, cerebrovascular disease and neuroinflammation could be especially relevant considering the well-known susceptibility of WM to these conditions Cerebrovascular pathology and chronic WM inflammation has been observed in animal models of different conditions known to increase the risk of dementia: global cerebral ischemia (mimicking heart failure), aging, obesity, hypercholesterolemia, type 2 diabetes, and diffuse axonal damage after TBI Interestingly, all the above-mentioned animal models of risk factors of AD have reported the chronic activation of a subset of microglia expressing Galectin-3  in the WM. Increased Gal-3 positive microglia has been associated with cognitive deficits in some models, for example, in aged rhesus monkey or hypercholesterolemic and diabetic mice ,154,155.Gal-3 is a β-galactoside-binding lectin that, presumably, promotes myelin-phagocytosis in MAC-1-activated microglia On the other hand, some groups have proposed that Gal-3 microglia activation follows oligodendrocyte or myelin abnormalities, and play a role in myelin sheath reparation by releasing nerve growth factor (NGF) and phagocytosing the degenerated myelin ,166. NGFNTR receptor, which mediates its trophic effects within the brain, while proNGF has a similar affinity for TrkA/p75NTR receptor and sortilin/p75NTR complex, the latter mediating the apoptotic pathway of the neurotrophin . Under pathological conditions, an imbalance in TrkA vs p75NTR receptor ratios increases the sortilin/p75NTR complex formation responsible for the proNGF switch from neurotrophic to apoptotic activity .7Changes in lifestyle, early detection of CVRFs in midlife populations, and therapeutic interventions targeting the brain vasculature, therefore ensuring healthy hemodynamics, may constitute effective avenues for the clinical management of populations at high risk of suffering from late-onset AD with a vascular component.Understanding what triggers WM changes, and the link with vascular pathology, will shed light on the relationship between the cerebrovascular dysfunction seen in many risk factors of AD and the neurodegeneration finally leading to cognitive decline. Namely, early detection of fornix degeneration in patients with CVRFs or TBI could help prevent the development of cognitive deficits or dementia later in life.On the other side, understanding what causes WM pathology in the fornix, and the cascade of events leading to WM dysfunction, could help the optimization of techniques such as DBS-f for the treatment of cognitive decline, as well as other pharmacological or nonpharmacological approaches aiming to halt WM degeneration. For this reason, more research is necessary to reveal the specific cellular and molecular mechanisms causing WM damage in risk factors of AD.María Lacalle-Aurioles: Writing – review & editing, Conceptualization. Yasser Iturria-Medina: Writing – review & editing.On behalf of all authors, the corresponding author states that there is no conflict of interest.


Abiotic stress is considered as the main culprit for reduction of global food production. Recent studies have reported GABA as a major regulator of abiotic stress and thus opening new avenues in research on emerging roles of GABA in abiotic stress acclimation in plants. Being an intermediate of nitrogen metabolism, GABA also contributes to primary and secondary metabolic pathways in plants.5 Several studies have provided evidences about the relationship of GABA and polyamines with different plant hormones such as abscisic acid, cytokinin, auxins, gibberellins and ethylene, etc., which are important for the plants facing different stress conditions. As per foregoing works, GABA has been found to be involved in enhancing the plant tolerance toward temperature,7 water scarcity,8 salinity,9 low light and nitrogen starvation.10 The role of GABA regarding single stress effect is sufficiently available with adequate researches, whereas its effects during combined stress on plants are not enough. Works on stress combinations of salt and drought, as well as salt, drought, and heat combined,11 are an example of GABA’s role in their regulation. One such experiment regarding combined stress has been performed by Balfagon et al.12 which is concomitant with GABA’s rescuing property but with different gene expressions in comparison to single stress subjection.A four-carbon well recognized, ubiquitous, non-proteinogenic amino acid, -aminobutyric acid (GABA) was initially discovered as a metabolite in plants during the mid-20viz. the GABA shunt pathway and generation through the breakdown of polyamines (Pas).12Though, GABA accumulates aggressively under stressed cellular milieu, it is essentially present in all the plants analyzed so far under optimum concentrations for proper plant growth. There are two known pathways of its endogenous biosynthesis, 12 exposed Arabidopsis thaliana Col-0 and its mutants to elevated light intensity (HL), heat stress (HS) and their combination (HL+HS), as it is both the stresses are seldom separable in the environment. They did the relative analysis of stressors on mutants of GABA biosynthesis , gad1-5 (CS860069)), and autophagy impaired mutants  in order to analyze the metabolic accumulates and degenerates, physiological responses and their mitigation by GABA in coordination with autophagy.Balfagón et al.The authors interestingly reported that HL+HS facing Col-0 plants show an upsurge in the transpiration and stomatal conductance rather than stomatal closure related with minimizing loss of water during stress along with the limited rate of photosynthesis, suggestive of strategy to rescue antenna complex and simultaneously cooling down of the leaf temperature. Furthermore, the metabolite analysis using gas chromatography and TOF mass spectrometer specifically display a heightened increase in the level of GABA along with glycerol, succinic acid, rhamnose, arginine, gluconic acid, tyrosine, under HL+HS justifying its significance in mitigating amalgamated stress. Furthermore, glucose, fructose, maltose and raffinose get accumulated in HL+HS along with slight increase in sucrose, conclusive of the increased photosynthetic accumulates due to the increased energy demand and HL. Alternatively, the assay of the TCA cycle transcriptome and metabolome displays diminishing metabolite levels, particularly aspartate and glutamate with high rate of TCA-related enzymes expression, indicative of imbalance of TCA cycle where the throughput genic expression was high but with low functional metabolites.gad3 mutant lines, when examined under HL+HS followed by a sharp decline in the survival rate percentage and increased leaf damage index along with extremely low rate of photosynthesis, transpiring rate and stomatal conductance, and damaging increment in the leaf temperatures. These results established that GABA is intimately involved and essential for the rescue of plants stressed with HL+HS. While in the case of individual stressors either HL or HS, the presence of GABA remains unnoticeable. Using RNA sequencing data, analysis of transcript expression involves in GABA biosynthesis and GABA catabolism shows remarkable increase in GAD1 and GAD3 (the GABA biosynthetic transcripts), whereas there is decrease in expression of POP2 and ALDH5F1 (the GABA catabolic transcripts) observed in HL+HS. The minor recovery noticed in gad3 may be subjected to the action and presence of GAD1, synthesizing GABA in miniscule amounts as GAD1 displays a 4.64% and GAD3 displays 29.03% enhancement in Col-0 under HL+HS. Reduced glutamate and putrescine levels signify the GABA generation through both its biosynthetic pathways, i.e., GABA shunt and oxidation of polyamines. Abiotic stress causes putrescine oxidation which results in GABA production; this signifies that reduction in putrescine level during combined stress causes accumulation of GABA in plants.In contrast to Col-0, the absence or declining of GABA is observed in atg(5 and 9) mutants while survival rate under HL or HS continued to be nearly unaffected in atg plants showing minor leaf damage but not death under HL. Along with the recent reports, this result overlaps with the fact that the triggers of GABA and autophagy-related genes under HL+HS might be interlinked and tend to acclimatize the plant against stress by decreasing the leaf temperatures through stomatal conductance and increasing transpiration thereby suppressing the leaf death index.Interestingly, a similar observation is to be made for the phenomenon of autophagy, where Col-0 plants show enhanced resistance to HL+HS in comparison to 13 It would be interesting to study the role of GABA biosynthesis , GABA catabolism transcripts (ALDEHYDE DEHYDROGENASE 5F1 [ALDH5F1] and POLLEN-PISTIL INCOMPATIBILITY 2 [POP2])14 expression under different combination of interrelated stresses of nature, like drought often accompanied with high heat and high light (GAD1/2 has been seen to function in rescue utilizing GABA under drought15 and salinity stress.16 Therefore, it would be intriguing to see which one of the known transcripts and metabolites get involved in such stress combination as reported by Balfagón et al. 12 and Shaar-Moshe et al.11 or if other unique transcripts partake during the process thereby bringing research closer to formulate strategies for better adapted plants against stress combination. Further work on amalgamated stress may provide various unknown aspects of GABA which are speculated to deal with abiotic stress in precise manner. It would be an interesting arena for the researchers to formulate strategies for the plants for their better adaptation to abiotic stresses on global level.In conclusion, the idea of studying two interrelated stresses and combining them makes the experimental setup closer to the natural growth conditions. HL+HS stress combination displays morphological, physiological and metabolomic variations with respect to individual HL or HS, with GABA acting as a rescuer of them. Exogenous application of GABA has been reported to improve the plant tolerance to different stresses. It is being evident that GABA enhances expression of certain heat shock factors, causing the plants to become heat tolerable.gh light . Drought


The Research Topic Cell Transplantation in Diabetes: Cell-Based Immunotherapy for Type 1 Diabetes, represents a collection of review articles, mini review articles and original research articles, which together describe the research status, latest strategies and development direction of islet transplantation and stem cells in type 1 diabetes (T1D) treatment.Type 1 diabetes mellitus (T1D) is an autoimmune disease characterized by absolute insulin deficiency caused by pancreatic β-cell-specific immune damage . It is aKoehler et al. reviewed the immunomodulatory and anti-inflammatory properties of MSCs in islet transplantation. Some challenges currently faced by islet xenotransplantation are also presented. Neonatal or young pigs could provide higher islet yield than adult pigs. The main disadvantage of porcine pancreatic islet cell clusters (ICCs)  was the lack of integrity and maturity. Porcine MSCs could improve islet function of ICCs and graft survival after transplantation. Microencapsulation technology could address the need for immunosuppression and protect islets from immune attack while still enabling the exchange of oxygen, insulin and nutrients. However, delayed and impaired transplantation prognosis and the formation of encapsulated fibrosis due to immature islet cells still severely limited the clinical application of encapsulated islet transplantation.Islet transplantation has become an effective method for the radical treatment of advanced diabetes due to the advancement of isolation technology and transplantation protocols. However, there is a severe shortage of pancreatic donors suitable for islet transplantation, which limits its wide clinical application. Porcine islet xenotransplantation is a promising approach to overcome the bottleneck of islet therapy in T1D. However, the delayed insulin secretion caused by the immaturity and immunogenicity of neonatal porcine islets remains a challenge for its clinical application. In the field of islet transplantation, numerous studies have shown that mesenchymal stem cells (MSCs) are used to improve islet function and graft survival after transplantation. The mini review article by Pomposelli et al. introduced a new strategy for composite islet-kidney allo-transplantation for the simultaneous treatment of T1D and diabetic nephropathy. At present, most of the reports chose pancreatic islets to be transplanted into the liver through the portal vein. This site was rich in blood supply, and insulin secretion and release from the portal vein system were more in line with the physiological pathway of insulin, but it may cause serious complications such as portal hypertension and liver failure. Combined islet-kidney transplantation allowed islets to be transplanted under the renal capsule in advance, allowing them to be prevascularized in the host environment. This not only avoided instant blood-mediated inflammatory reaction (IBMIR) in the early stages of transplantation, but also provided a persistent physical barrier to the host’s innate immune response. In addition, a new strategy was to incubate islets with anti-inflammatory nanoparticles to reduce islet cell apoptosis after transplantation, which could reduce the number of donors required. It also solved the defect that only a limited volume of islet grafts can be accommodated under the renal capsule. By using a durable chimera strategy to induce tolerance in the host, transplant rejection can be avoided, thereby avoiding the long-term use of immunosuppressive drugs, and avoiding the side effects of immunosuppressive drugs. Moreover, placing the islet graft under the renal capsule made subsequent harvesting of the graft relatively easy, facilitating observation and exploration. Animal experiments of islet-kidney combined transplantation suggested that this strategy was an effective means for the treatment of T1D and end-stage renal disease. In order to further improve the clinical effect of this scheme, especially the function of transplanted islets, it needed to be used in combination with other strategies. Moreover, islet-kidney combined transplantation was still in the exploratory stage of animal experiments, and the long-term efficacy of clinical application needed to be further evaluated.When the islets were separated from the pancreatic tissue, the blood supply of the islets was destroyed. At that time and at the initial stage of transplantation, the islets only relied on the diffusion of oxygen in the surrounding environment to maintain their survival. Therefore, the oxygen partial pressure in the center of the islet was extremely low, resulting in the occurrence of cell death and apoptosis in the center of the islet. The revascularization of islets after transplantation was slow, and even after revascularization of the transplanted islets, their blood supply and partial pressure of oxygen were still significantly lower than those in normal pancreas. Therefore, accelerating the angiogenesis around islets after transplantation and increasing the density of blood vessels through corresponding strategies have become the focus of recent research in the field of islet transplantation. The mini review article by in vivo are important for understanding the underlying causes of graft failure, reflecting the quality of successfully transplanted islets, as well as islet survival and insulin secretion in vivo. A review by Arifin and Bulte described how imaging techniques can be used to trace and monitor islet graft distribution, number or volume, viability, and function in vivo. They outlined bioluminescence (BLI), fluorescence microscopy imaging (FMI), Single-photon emission computed tomography (SPECT), Positron emission tomography (PET), magnetic resonance imaging (MRI), magnetic particle imaging (MPI) and ultrasound imaging, the advantages, limitations and clinical utility of each specific imaging method.In addition to the limitation of donor organ shortage, islet transplantation therapy is also affected by autoimmune and allogeneic rejection as well as non-immune-related factors. Non-invasive techniques for monitoring and assessing islet graft fate in vivo islet imaging, and much work remained to be done to further improve the accuracy and efficacy of image-guided islet therapy.Currently, BLI and FMI had been widely used in the evaluation of transplanted pancreatic islets in animal models, but they were not suitable for clinical patient studies due to their invasiveness or lack of light penetration in deep tissues. PET, SPECT, MRI, MPI, and ultrasound were currently being developed for this specific purpose, and unlike BLI or FMI, they had great potential for clinical translation. For pancreatic islet imaging technology to be successfully applied in clinic, the labeling technology needed to achieve low toxicity of the marker, long and stable labeling time, and high detection sensitivity. Recent progress had been made in the field of Zhang et al. conducted a retrospective cohort study of 11 patients with immune checkpoint inhibitor-induced type 1 diabetes (ICIT1D) in their original research article. In the study, they found that among patients with autoimmune diabetes, 25% (2/8) had elevated lipase levels and no patients (0/6) had elevated amylase levels. Combined with longitudinal clinical studies, the results suggested that ICI can induce selective pancreatic endocrine or exocrine toxicity. Studies suggested that GAD65 antibody was the most sensitive autoantibody associated with ICIT1D, but should not be used as a necessary criterion for diagnosis. The study also described various clinical manifestations of ICIT1D, with late onset age, rapid and significant pancreatic islet failure, and concurrent of other gland damages. Therefore, improving the understanding and attention of domestic doctors and patients to ICIT1D, early identification and timely initiation of insulin therapy may avoid serious complications of autoimmune diabetes, such as diabetic ketoacidosis (DKA).Diabetes mellitus induced by immune checkpoint inhibitor (ICI) therapy is a rare but potentially life-threatening complication. It is unclear if ICI treatment causes selective islet toxicity or non-selective pancreas toxicity, for the purpose of answering this question, in vivo setting have not been systematically studied and demonstrated. It is well known that human β cells express human leukocyte antigen class I (HLA class I), and HLA class I is involved in the pathogenesis of T1D by expressing diabetogenic antigens to CD8 T cells. To study the interaction of human sBCs and T cells, a reproducible, well-defined in vitro assay system was established to measure the stimulation of sBCs by human autologous CD8 T cells in the original research article by Castro-Gutierrez et al. Specifically, the surface expression of HLA class I of sBC was knocked out using genome engineering, and the inducible overexpression of the immune checkpoint inhibitor programmed death ligand 1 (PD-L1) was integrated. The results showed that sBC stimulated human diabetic CD8 T cells in an HLA-dependent manner, and PD-L1 overexpression could effectively reduce this stimulation. This suggested that manipulation of the HLA class I and PD-L1 receptor could provide protection in humans against diabetes-specific immune recognition.Studies using preclinical animal models have demonstrated that transplantation of stem cell derived beta-like cells (sBCs) can rescue diabetes. However, the safety and efficacy of sBCs for clinical diabetes treatment and how sBCs respond to the inflammatory milieu of diabetic T cells in a rigorous human In conclusion, the studies outlined in this Research Topic demonstrate that cell therapy focuses on two core mechanisms of T1D development and exerts therapeutic effects on islet β-like cell replacement or immunomodulation. Islet transplantation directly achieves islet function reconstruction at the cellular level. Pluripotent stem cells provide an important source of cells for islet transplantation by inducing differentiation into islet β-like cells. MSCs can provide a favorable internal environment for the survival of β-like cells by inducing immune tolerance and immune regulation. The combination of different types of cell therapies may complement each other in terms of mechanisms, thereby improving efficacy, curbing or even reversing disease progression.All the authors have made a substantial, direct and intellectual contribution to the work and approved it for publication.The authors declare that this editorial was prepared in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Rye is the only cross-pollinating small-grain cereal. The unique reproduction biology results in an exceptional complexity concerning genetic improvement of rye by breeding. Rye is a close relative of wheat and has a strong adaptation potential that refers to its mating system, making this overlooked cereal readily adjustable to a changing environment. Rye breeding addresses the emerging challenges of food security associated with climate change. The systematic identification, management, and use of its valuable natural diversity became a feasible option in outbreeding rye only following the establishment of hybrid breeding late in the 20th century. In this article, we review the most recent technological advances to improve yield and yield stability in winter rye. Based on recently released reference genome sequences, SMART breeding approaches are described to counterbalance undesired linkage drag effects of major restorer genes on grain yield. We present the development of gibberellin-sensitive semidwarf hybrids as a novel plant breeding innovation based on an approach that is different from current methods of increasing productivity in rye and wheat. Breeding of new rye cultivars with improved performance and resilience is indispensable for a renaissance of this healthy minor cereal as a homogeneous commodity with cultural relevance in Europe that allows for comparatively smooth but substantial complementation of wheat with rye-based diets, supporting the necessary restoration of the balance between human action and nature. Secale cereale L.) entered its European growth habitat as a weedy grass in wheat/barley agricultural systems and evolved as a secondary crop by initially mimicking the visual appearance of Triticum spp. and Hordeum vulgare [Rye , the staple crop for an estimated 35% of the human population, which will continue to play a critical role to ensure an adequate and affordable intake of calories and proteins in diets until 2050 [T. aestivum approximately 3–4 million years ago [In recent decades, European agriculture has experienced higher, more pronounced and more widespread climate variability as compared to the long-term average . In 2022til 2050 . Rye divears ago  and is cears ago ,13. The ears ago . In factears ago . These rears ago  to a chaThe systematic identification, management, and use of valuable natural diversity in outbreeding rye only became a feasible option following the establishment of hybrid breeding late in the 20th century. The rationale and main driver for hybrid breeding is the systematic exploitation of economic or standard heterosis that refers to the superiority of a hybrid over standard commercial check varieties. In rye, hybrid breeding is a success story in terms of the selection of favorable alleles for grain yield. In sharp contrast to the competitiveness of hybrid compared to line breeding in wheat , the keyper se performance and is designed to measure alterations in highly heritable traits such as plant height, thousand-grain weight, and disease resistance with high sensitivity  systems ,46,47,48Both commercially used CMS systems differ in their impact on hybrid rye breeding. The G cytoplasm belongs to a plasmotype that is common in the Central European rye gene pool . Its invRf) genes. Major Rf genes enabling grain production in CMS-based hybrids are available for both the G and the P cytoplasm, and have been mapped on the distal end of the long arm of chromosome 4R [Rfp genes originating from non-adapted genetic resources is mainly monogenic dominant-inherited [Rf genes on the short arm of chromosome 1RS and the long arm of chromosome 3R [In hybrid progenies from selective mating, the CMS effect of the seed-parent genotype needs to be counteracted by nuclear alleles from the pollen parent that restore pollen production. Such genetic factors, thus, are designated restorer-of-fertility  Tulasne, the species of greatest concern [Ergot is a disease of cereals and grasses caused by fungi in the genus  concern . At anthC. purpurea virulence factors have been reported, RNAseq analysis of in planta-expressed fungal genes identified more than 400 highly expressed transcripts including an elevated frequency of genes encoding putative effectors that might be involved in repelling the fungal attack or interfering with host-defense interactions [Rf genes is the most effective approach for minimizing ergot infestation in hybrid rye. Because of the toxicity of ergot alkaloids for humans and animals, the European Commission has significantly lowered the thresholds for ergot since 1 January 2022. Supplementary to the previous threshold of 0.05% ergot sclerotia in unprocessed rye, harvested grain may not contain more than 500 µg/kg of total ergot alkaloids in rye milling products for human consumption [Noteworthy in this context is a protective effect provided by ergot on cereal rye that probably acted against human exploitation, as heavily affected seed and grain samples have been, and still are, disposed of. This example of conditional host-pathogen interaction in which the consequence may fall on different parts of a continuum from parasitism to mutualism depending on the specific situation might explain why grasses are commonly susceptible and rather tolerant to ergot and no effective resistance mechanisms have evolved over time . Indeed,ractions . Althougractions , specifisumption . From 1 Rfp1 only [Rfp1 originates from a weedy rye population from the Fertile Crescent [Rfp1 in rye have been reported influencing seed development through male gametogenesis or post-fertilization, due to factors deposited by the sperm cells in the egg and/or central cells, as well as by genomic imprinting [Arabidopsis thaliana, with a much larger influence of the paternal genome on F1 seed size than previously appreciated [1 seed size has been observed in this study as well. These results from fundamental research have implications for breeding research on the seed biology of hybrid rye and open new avenues for genetic approaches to compensate for paternal contributions to seed size in rye hybrids with strong ergot defense. Susceptibility to ergot upon artificial inoculation is a trait assessed for registered rye varieties since 2008 in the descriptive variety list of the German Federal Plant Variety Office. The description of rye varieties listed in 2022 for their resistance and yield traits demonstrates genetic gain for ergot defense in highly productive hybrids already at the level of open-pollinating cultivars . Howeverfp1 only , the devCrescent ,65,70,78Crescent . This reCrescent . Paternaprinting . Recentlreciated . NotablyRfp genes while simultaneously minimising the costs of restoration in terms of grain yield. Particularly promising for this purpose are genes that cause parent-of-origin effects on F1 seed size such as MET1, the primary maintenance DNA methyltransferase in Arabidopsis thaliana. Crosses between Arabidopsis genotypes carrying a complete loss-of-function and the wild-type MET1 allele revealed that hypomethylated maternal genomes result in significantly larger F1 seeds [SECCE2Rv1G0093370 [MET1. In total, 6 SNP markers on the rye 600k SNP-array [SECCE2Rv1G0093370 between elite inbred lines and germplasm collections. Subsequently, an ingenious cross-validation strategy [SECCE2Rv1G0093370. Superior SNP alleles leading to hypomethylated maternal genomes as measured by significantly larger F1 seeds are attractive means to offset the undesired paternal inherited effects of Rfp1.Our steadily growing knowledge of the genetic code of grain size and weight control ,82 and kF1 seeds . In rye,G0093370  shares 5NP-array  provide This drafted option for selection with markers and advanced reproductive technology (SMART) breeding  in rye eRfp1 on grain yield but may result in insufficient quantities of pollen to combat the fungus adequately in farmers’ fields, as wet pollen agglutinates and distributes over short distances only. In fact, multi-environmental data on natural ergot infection provided by the Polish Research Centre for Cultivar Testing (COBORU) confirm this assumption  of them become available. The current breeding scheme is two generations shorter than the standard scheme [per se performance are selected from the base population and crossed to two heterozygous testers representing the ‘Carsten’ and ‘Petkus’ pool. The testcrosses are subsequently evaluated in 3–4 target environments with respect to their yield performance to identify the GCA of the selected genotypes  . RRS is  testers . A greatd scheme , and thuenotypes D. Based 2 are used to establish further testcrosses to achieve robust performance data on yield potential, stability, and adaptation with the second GCA test in 8 to 10 highly divergent environments in terms of soil fertility and precipitation . Recently published reference genome sequences ,42 and hKnowledge of genomic regions controlling complex traits is the key to our understanding of mechanisms behind yield architecture and to using them in marker-assisted crop improvement programs. The novel catalog of SNP variations associated with yield and grain weight holds potential for the clear-cut and systematic genetic improvement of yield potential in rye hybrids with strong ergot defense. Hybrid breeding offers a pragmatic strategy to evaluate the influence of single genomic loci on grain yield in rye. Progenies segregating for superior (S) or inferior (I) SNP alleles can be used to establish near-isogenic genotype bulks (NIB) of homozygous elite F3 lines of the seed-parent pool, that will serve as pollinators in crosses with male-sterile single-cross testers from the opposite pool between isolation walls . In the Although the development of perfect markers that address the functional sequence polymorphism of a gene governing grain yield would be the consistent final step, it needs to be considered that the necessary functional characterization of a candidate gene by transposon tagging, transformation for overexpression, knockdown analysis, or genetic modification based on novel plant breeding techniques is still in its infancy, i.e. not available or published in rye. The extension of the molecular toolbox available for rye by sophisticated technologies probably is a long-term perspective. In contrast, the proposed International Functional Genomics Project on Rye (IFGPR) for the development of highly replicable rye genetic stocks in the public domain  appears Dominant dwarf 1 (Ddw1), identified five decades ago by Vladimir Kobylyansky in the germplasm collection at the N.I. Vavilov Institute of Plant Genetic Resources [Estestvennyj Mutant 1 , has been used in breeding of open pollinating cultivars for the genetic improvement of plant height [Ddw1 has not been used in hybrid breeding programs due to theoretical considerations [The introgression of major dwarfing genes for increasing yield potential is a proven concept in wheat and rice ,130,131.esources  and initt height , a quantt height ,134,135.t height ,138,139.t height ,140,141 erations ,134,135.erations . Ddw1 in hybrid rye, novel markers [Ddw1 into the seed-parent pool. Homozygous semidwarf BC3S2 single-cross testers enabled the development of semidwarf P-type hybrids in 2020 for the first time ever. Subsequently conducted performance trials in European and Canadian target environments for rye cultivation revealed that the semidwarfs were distinct from existing conventional hybrid rye varieties and expressed the genetically reduced plant height uniformly , the semidwarfs revealed a pronounced lodging resistance as a result of their shorter and sturdier stems . In viewproducts . SemidwaDdw1 belongs to the group of gibberellin (GA)-sensitive dwarfing genes [Eragrostis tef (Zucc.) Trotter) and finger millet (Eleusine coracana Gaertn) have been reported as a result of chemically induced GA deficiency [GA2ox2/6 and an increased drought tolerance compared to the wild-type plants. In semidwarf rye the gene ScGA2ox12 co-segregating with the GA-sensitive dwarfing gene Ddw1 is up-regulated as well [Ddw1 mutant alters the GA content in a favourable manner. Notably, the integration of the Ddw1 target interval in the wheat reference genome sequence indicated perfect micro-colinearity between the Ddw1 locus and a 831 kb segment on chromosome 5A, which resides inside of a 11.21 Mb interval carrying the GA-sensitive dwarfing gene Rht12 in wheat [Rht12 overgrowth mutants with a range of intermediate height phenotypes taller than the original Rht12 stock [ng genes . Noteworng genes . Positivficiency . In riceficiency . In accoficiency . The ect as well , supportin wheat . The kno12 stock  as novelDdw1 on the line per se performance in thousand-grain weight and flowering time have been reported [Ddw1 on the testcross performance of genetically divers rye hybrids, and in a reasonable number of environments, were lacking until recently. Indeed, extensive phenotyping of conventional experimental hybrids representing a high level of genetic diversity in two years with contrasting precipitation patterns improved our understanding of rye’s adaptation to drought stress [Ddw1 as an option to tap currently unused potential for optimized dry matter allocation to the rye grain. However, it may be misleading to transfer knowledge from conventional to semidwarf genotypes without empirical data. For instance, in semidwarf Ddw1 genotypes the head has proven to be supplied with assimilates essentially via the flag leaf (F) and F-1 [Ddw1 on the performance of rye in target environments of rye cultivation. The enhanced implementation of Ddw1 in hybrid rye breeding serves as a viable example of breeders’ options to target allelic combinations of genes controlling phenology and plant architecture. In this way, further known major genes such as VRN, PPD, EPS [Concerns have been raised about a potentially negative impact of genetically reduced culm length in rye on photosynthesis and yield performance ,135,142.reported . Howevert stress . This co and F-1 , in cont and F-1 . The comPPD, EPS , and genPPD, EPS  are wortDdw1 in a practical hybrid rye breeding program is paradigmatic for specific genes and adaptive alleles that govern important agronomic traits in rye. The promising performance in target environments of rye cultivation triggers an enhanced development of semidwarf hybrids and may initiate a new era of physiological rye breeding that aims to raise the yield potential of rye closer to its biological limit. Indeed, the rich genetic diversity of rye, together with a sophisticated bulked segregant phenotyping strategy of testcross performance in multi-environmental field trials, argue for a stronger utilization of rye in research directed to the identification of valuable alleles controlling yield and yield components for Triticeae improvement programs. For this purpose, further investment in resequencing is promising for an improved understanding of the genetics of adaptive traits in rye.Despite current achievements, major challenges in rye production remain for advancing rye from its current status as a healthy minor cereal to a trend-setting crop with increasing relevance for grain production in a changing climate. Therefore, improved breeding efforts are strategically important for enhancing the competitiveness of rye in agricultural production systems. In rye, hybrid breeding serves as a cutting edge technology to select crossing partners on a rational basis for breeding improved cultivars with different complex but complementary traits to achieve cumulative gene action for grain yield and yield stability. The identification of traits that may allow rye to capture its most limiting resources more efficiently receives strong support from two high-quality rye genome assemblies ,42 and pHybrid breeding had obvious advantages and positive side effects in rye. In contrast to many valuable crops with a relatively small acreage that became orphans for which breeding was no longer carried out , hybrid


The work producedby Gomes et al., with the objective ofreducing the spreading of the infection and its inherent complications.The division between groups using the surgery-infection time relationship allows us todelineate the outcomes presented. The high mortality identified in groups 2 and 3corroborates the data described in the literature. In addition to this finding, theprevalence of acute renal failure is highlighted, as well as the longer hospital staybetween the groups. The changes and protocols initiated by several surgical services forthe care of COVID-19 patients were important in the pandemic, as described by Casanovaet al., in a descriptiveanalysis of 97 patients in which 7 of these individuals were diagnosed with COVID-19infection in the immediate postoperative period, underwent the following procedures:coronary artery bypass grafting , aortic valve replacement (n=1), aorticvalve replacement + CABG (n=1) and mitral valve replacement + CABG (n=1). The mortalityrate in this case series was 3 (43%) patients. The authors also emphasize that all 3deaths were due to respiratory failure refractory to the measures adopted by theassistant team.Yates et al., in a single-center analysis of 13 cases undergoing CABG surgery,identified 3 deaths. Patients with a positive PCR testing for SARS-CoV-2 before surgerywaited 14 days for symptoms to resolve according to institutional protocol. Two of thesedeaths were due to septic shock and encephalopathy. Three patients underwent emergencyCABG, therefore with unavailable previous exams, of which 1 patient evolved withcardiogenic shock and death on the 3rd postoperative day. In addition tothese findings, the patients had a long hospital stay as well as other complications,such as renal failure and pulmonary thromboembolism.In another case series, Farsky et al. that there is high mortality and morbidity associated withCOVID-19 infection in patients undergoing cardiovascular surgery Longer hospital stay,pulmonary complications, renal failure and thromboembolic events are present in thesepatients. Preoperative management protocols and increasingly robust scientific data tounderstand the evolution of patients between different times of infection are thedirection in this time of uncertainty.Such studies corroborate the findings of Gomes et al.


Alcohol use disorder is a highly significant medical condition characterized by an impaired ability to stop or control alcohol use, compulsive alcohol seeking behavior, and withdrawal symptoms in the absence of alcohol. Understanding how alcohol modulates neurocircuitry critical for long term and binge-like alcohol use, such as the central amygdala (CeA), may lead to the development of novel therapeutic strategies to treat alcohol use disorder. In clinical studies, reduction in the volume of the amygdala has been linked with susceptibility to relapse to alcohol use. Preclinical studies have shown the involvement of the CeA in the effects of alcohol use, with lesions of the amygdala showing a reduction in alcohol drinking, and manipulations of cells in the CeA altering alcohol drinking. A great deal of work has shown that acute alcohol, as well as chronic alcohol exposure via intake or dependence models, alters glutamatergic and GABAergic transmission in the CeA. The CeA, however, contains heterogeneous cell populations and distinct subregional differences in neurocircuit architecture which may influence the mechanism by which alcohol modulates CeA function overall. The current review aimed to parse out the differences in alcohol effects on the medial and lateral subregions of the CeA, and what role neuroinflammatory cells and markers, the endocannabinoid system, and the most commonly studied neuropeptide systems play in mediating these effects. A better understanding of alcohol effects on CeA subregional cell type and neurocircuit function may lead to development of more selective pharmacological interventions for alcohol use disorder. Alcohol use disorder (AUD) is a medical condition affecting around 15 million individuals in the US annually, characterized by an impaired ability to stop or control alcohol use despite adverse social, occupational, or health consequences . Long tein vivo. As the CeA contains heterogeneous subregions, alcohol effects on CeA subregion neurocircuits are also heterogenous. For instance, some studies show an increase in glutamatergic transmission in the lateral subregion of the CeA following acute alcohol , medial (CeAM), and lateral capsular (CeAC) subdivisions   . These cs (BNST) . AlthougM, decreases glutamatergic transmission in the CeAM, and increases spontaneous glutamatergic transmission in the CeAL  concentration of alcohol, there was a significant increase in the spontaneous inhibitory event frequency compared to baseline in dependent, but not naïve, females. This suggests that acute alcohol application does not have any effect on inhibitory transmission at any concentration in alcohol naïve females and that chronic alcohol exposure induces mild sensitivity to the effects of acute alcohol  of GABAB receptors suppresses alcohol related behaviors in rodents  receptors, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, and metabotropic G protein coupled receptors (mGluRs). Studies have shown that acute alcohol reduces glutamatergic transmission in the CeAM through NMDAR and non-NMDAR mediated mechanisms by inhibiting glutamate release  rats – a strain of rats selected for their high alcohol preference – have a higher basal frequency of spontaneous excitatory postsynaptic currents compared to Wistar control rats  mediated by both presynaptic and postsynaptic mechanisms, (2) mediated by action potential dependent and independent mechanisms, (3) are strain specific, and (4) are dependent on the history of alcohol exposure (acute vs. chronic). The complexity of the mechanisms of action of alcohol speaks to the need of further studying the effect of alcohol on the CeAM through a variety of techniques to parse out the exact points of alcohol action.In addition to both strain differences and recording type, the duration of alcohol exposure plays a role in the effect of alcohol on glutamatergic transmission in the CeA. Acute alcohol has been shown to reduce glutamatergic activity through both an NMDAR and non-NMDAR mediated mechanism . ChronicL is less extensively examined. A study by L in alcohol naive male C57Bl6/J mice and saw an increase in presynaptic glutamatergic activity after acute alcohol application (5–100 mM) with an estimated IC50 of roughly 20 mM. Expanding on these findings, a recent study from our lab showed that acute bath application of 20 mM alcohol increased spontaneous action potential dependent glutamatergic signaling presynaptically in both male and female C57Bl/6J mice, with no sex differences found SorloxSTOPlox–L10–GFP mice fired more action potentials compared to male non-alcohol drinking mice, while female alcohol drinking mice had a non-significant reduction in action potential firing compared to female non-alcohol drinking mice  and exogenous cannabinoids  for treatment of AUD has increased. Phytocannabinoids and exogenous cannabinoids have been approved in some areas for medical treatment of mental health disorders commonly associated with AUD, such as anxiety disorders and post-traumatic stress disorder. Two highly studied cannabinoids are cannabidiol (CBD) – a non-euphorigenic phytocannabinoid – and delta9-tetrahydrocannabinol (THC) – the main euphorigenic phytocannabinoid. CBD can act as a non-competitive negative allosteric modulator of CB1R and CB2R, especially in the presence of THC, leading to a reduction of the psychoactive effects of THC . ObservaNeuropeptides play an important modulatory role throughout the brain and the nervous system in general. In the CeA specifically, neuropeptides are involved in all of the actions of the CeA, including stress and anxiety , pain N, addicticrh gene that is highly involved in stress and fear response and is heavily implicated in alcohol use disorder . CRF is a neuropeptide encoded from the disorder . CRF has release . Additio release . Furtherknockout , suggestthe CeAL . In msP the CeAL . Behaviothe CeAL . Stimulathe CeAL . Additiothe CeAL . Howeverthe CeAL . TherefoM with a few neurons in the CeAL (Neuropeptide Y (NPY) is highly expressed in various brain regions, including the amygdala , and is the CeAL . NPY hasthe CeAL , making the CeAL . NPY infthe CeAL . Additiothe CeAL . OverallAt the circuit level, NPY has been shown to prevent and reverse alcohol-induced increases in GABAergic transmission through a presynaptic mechanism mostly mediated by the Y2 receptor . Three cM (∼60%), although the CeAL has a large population of Nts neurons as well (∼35%)  alcohol consumption activates the Nts neurons in the CeAL, (2) optogenetic stimulation of the CeA Nts+ projections to parabrachial nucleus (PBN) promotes consumption of alcohol drinking, and (3) the ablation of Nts neurons in the CeA decreases alcohol consumption in two-bottle choice test (The largest population of neurotensin (Nts) neurons in the CeA is found in the CeAl (∼35%) . Nts coml (∼35%) . Nts neuice test . In addiice test  – and moice test . Activatice test . Even thNociceptin acts through the nociceptin/orphanin FQ Opioid Peptide Receptor (NOP) and can have effects on alcohol-related behaviors . Oral adIntracerebroventricular (ICV) injections of nociceptin in msP rats led to a significant increase in alcohol intake acutely but led to a progressive decrease in alcohol intake when administered subchronically within a 9 days period . SimilarM  and somatostatin (SST)  . These cin (SST) . Specifiehaviors .L cells overlapping with tachykinin 2 (TAC2) cells – cells present in the CeAL and CeAM that are necessary for fear learning  and high (300 nM) concentrations, but had a significantly sensitized effect to intermediate concentrations of substance P (30 and 100 nM), suggesting an inverted U concentration effect curve. Additionally, in naïve and dependent rats, the increase of sIPSC frequency after acute 44 mM alcohol application was sensitized after 100 nM substance P application, and this effect remained after alcohol withdrawal  are present in the amygdala, with NK-1Rs being most widely present in the CeA in CeAC . Substan in CeAC . Interes in CeAC , with an in CeAC . A recenthe CeAM , which rthe CeAM . Substanthdrawal . These fin vivo manipulations of the CeA neurons when assessing the interaction between CeA neuronal activation and behavioral outcomes, such as alcohol consumption. It is worth noting that viral or pharmacologic manipulations of the CeA in vivo are limited in their approach, as currently, intracranial injections are not typically able to target the specific subregions of the CeA. More selective viral and pharmacologic approaches will be needed in future studies. Below is a brief summary of some of the literature on the relationship between alcohol consumption and in vivo CeA neuronal activity.The electrophysiological studies point to the involvement of the CeA in alcohol effects, however, it is important to consider A recent study by in vivo electrophysiological methods to study the role of CRF neurons on binge-like alcohol consumption in CRF-Cre mice. The group showed that pre-lick activated CRF cells had a higher firing activity than other CRF cells, with a steadily increasing firing rate over the length of the drinking experiment and in later drinking sessions compared to earlier ones (Studies have shown that intra-CeA injections of a kappa opioid receptor antagonist reduces binge-like alcohol drinking during a 4-h drinking in the dark session . Data frier ones . Additioier ones . This daL, increasing activity of CeAL neurons resulting in enhanced GABAergic signaling in the CeAM. CRF expressing neurons can also express Nts, which have been shown to act on GABAergic transmission in the CeA. Other neuropeptides, like SST, PKCδ, and substance P, may also regulate alcohol effects on the microcircuits in the CeA. The interplay between these neuropeptides and neuromodulatory systems with the endocannabinoid system and neuroinflammatory signaling may also alter the alcohol effect on neurotransmission in the CeA and alcohol-directed behaviors.The differential effect of alcohol on CeA neurotransmission is likely due to the actions of the subregion specific neuromodulators, neuropeptides, and neuroimmune cells. It is likely that the CRF1R and CRF2R modulate glutamatergic transmission in the CeAOverall, there are still many unknowns about the circuitry of the CeA and the role of specific various neuronal and non-neuronal cells and peptide systems in alcohol-related behaviors. Continued studies on intra-CeA microcircuitry as well as connectivity between the CeA and other brain regions may lead to novel therapeutic methods for treating AUD.MM researched and reviewed the cited articles and wrote the manuscript. YS worked with MM to make conclusions based on existing literature, create the figures, and edited the manuscript. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Arabidopsis thaliana since most of the relevant knowledge derives from this model organism. We will then proceed to provide perspective to what extent the relevant molecular mechanisms are conserved in other plant species and close by discussing current knowledge of the transcriptional machinery responsible for controlling the adaptive responses using selected examples.Plant cell walls are highly dynamic and chemically complex structures surrounding all plant cells. They provide structural support, protection from both abiotic and biotic stress as well as ensure containment of turgor. Recently evidence has accumulated that a dedicated mechanism exists in plants, which is monitoring the functional integrity of cell walls and initiates adaptive responses to maintain integrity in case it is impaired during growth, development or exposure to biotic and abiotic stress. The available evidence indicates that detection of impairment involves mechano-perception, while reactive oxygen species and phytohormone-based signaling processes play key roles in translating signals generated and regulating adaptive responses. More recently it has also become obvious that the mechanisms mediating cell wall integrity maintenance and pattern triggered immunity are interacting with each other to modulate the adaptive responses to biotic stress and cell wall integrity impairment. Here we will review initially our current knowledge regarding the mode of action of the maintenance mechanism, discuss mechanisms mediating responses to biotic stresses and highlight how both mechanisms may modulate adaptive responses. This first part will be focused on  Cell walls are essential for plant development and tightly involved in many signaling processes. They provide mechanical support required for land plants growing on soil and are normally the first barriers involved in perception of stimuli and activation of signaling pathways inducing responses in the plasma membrane, cytoskeleton, cytoplasm, and organelles. Relevant stimuli during interactions between plants and their environment can have either a physical or chemical nature  maintenance mechanism . Nevertheless, an increasing number of studies has started to shed light on the similarities and differences of CWI maintenance components in other plant species, including ferns, crops, and trees. Understanding how CWI functions in different plants species could have big economic impacts on plant-derived food and biofuel production  and Wall-associated kinase (WAK) subfamilies, which seem to interact with pectin via their extracellular domains , illustrates this difficulty. FER has been implicated in many different biological processes including female fertility, cell elongation, immunity responses and mechanosensing Höfte, . The rolCrRLK1L gene subfamily, was identified as a suppressor of the short-hypocotyl phenotype observed in the cellulose-deficient procuste1-1 (pcr1-1) mutant  show no phenotypic defects in non-stressed conditions, suggesting that THE1 becomes active only if CWI has been impaired, supporting the notion that it functions as CWI sensor. The the1-4 allele contains a T-DNA insertion in the region of the protein connecting the transmembrane and the kinase domains  prevent gene expression in the1-3 but not the1-4, thus making the1-4 a gain-of-function allele. Furthermore, THE1 is required for cellulose-deficiency-induced lignin accumulation —COMPLEMENTING ACTIVITY 1 (MCA1) and the RLK FEI2 regulating CWI , the founder member of the the1 seedlings and plants only exhibit a visible phenotype when CWI is impaired  result in diverse and often strong mutant phenotypes. This is particularly surprising since the majority of the family members have broad expression domains and their expression is activated by specific abiotic and biotic stresses , their knockout phenotypes are drastically different. FER knock down allele leads to enhanced production of JA, SA and lignin after exposure of fer-5 seedlings to CWD, similar to the effects of the the1-4 gain of function allele  loss-of-function allele, suggesting that de-esterified pectin activates the WAK2 hyperactive allele. -containing extracellular domain, a transmembrane region and a conserved kinase domain Kohorn . Arabido2+-DEPENDENT PROTEIN KINASES (CDPKs), modulation of hormone-based signaling, reorganization of the cytoskeleton and changes in gene expression . However, a minor strategy involves recognition of fungal chitin by LYSIN MOTIF-CONTAINING RECEPTOR-LIKE KINASE 5 (LYK5) and CERK1  leading to ROS production and activation of the MAPK signaling module  form another relevant group of PAMPs  are well-established DAMPs and play an important role in PTI  result in activation of PTI in a similar manner as OGs  MYC2 influencing JA signaling  supergene family and function as transcriptional activators of CESA1, CESA3 and CESA6, which are involved in primary (but not secondary) cell wall formation , lacking secondary cell walls, produced multilayered, thickened fiber cell walls without ultra violet autofluorescence indicative for the presence lignin, therefore resembling primary cell walls  TFs VND7 expression, in agreement with previous reports showing that E2FC can act as a transcriptional activator and repressor , POLYGALACTURONASE INVOLVED IN EXPANSION 3 (PGX3), and MYB DOMAIN PROTEIN 54 (MYB54) in response to salt stress  genes play an essential role during fruit ripening and their expression is regulated by histone modifications such as H3K9/K14 acetylation and H3K27 tri-methylation  modulates directly expression of genes required for cell wall loosening such as CrRLK1L kinase signaling pathway is specific for land plants, since analysis of algae genomes identified neither orthologues for CrRLK1 members nor for RALFs peptides  found in Marchantia polymorpha, exhibits the highest similarity to FER from Arabidopsis, highlighting again its particular importance (despite having been referred to as MpTHE before) . Based on the expression patterns of several legume CrRLK1Ls they have been implicated in root nodulation.A comprehensive genome-wide comparative analysis of the Brachypodium distachyon (hereafter Brachypodium) in rapidly growing tissue implicates them in cell expansion  is provided by WAKL4, also named Stb6, through recognition of apoplastic avirulent effectors , the number has increased to 29 in cotton, 125 in rice, 341 in wheat, more than 100 in maize, 91 in Barley and 115 in CrRLK1L FERONIA family in rice has 12 FERONIA-LIKE RECEPTORS (OsFLR) members , where transient silencing of TaFER1 and TaFER2 had no obvious impact on Fusarium graminearum infection , which afterward binds to (OsCERK1) whereas in Arabidopsis, AtCERK1 binds chitin directly. The OsCEBiP/OsCERK1 complex in turn activates signaling components such as OsRacGEF ) through a MAPK-signaling module  and RECEPTOR-LIKE CYTOPLASMIC KINASE 176 (OsRLCK176) are also positive regulators of immune responses in rice and interact directly with the OsCEBiP/OsCERK1 complex  in Arabidopsis, and 173 members  in rice  lead to enhanced resistance against the bacterial blight (Xanthomonas oryzae pv. oryzae-Xoo), increased cell wall strength while reducing plant height to a small extent and not interfering with grain yield  and NAC31 to repress cell expansion and wall thickness. Furthermore, the cotton (Gossypium hirsutum) gene KNOTTED1-LIKE, member of the GhKNOX family, regulates fiber development, initiation and elongation possibly by forming heterodimers with OVATE FAMILY PROTEIN 4 , the NAC TF EgNAC141 activates genes required for lignin biosynthesis  and OsCESA7  and ZmGCN5, which can result in increased acetylation of H3K9 on at histone H4K5  and XYLOGLUCAN ENDOTRANSGLYCOSYLASE (ZmXET1) genes  homologs, gene silencing (Hu et al. Epigenetic regulation in dicots and especially in AtFER function has been characterized extensively mainly because of the pleiotropic effects observed in the loss-of-function mutants, indicating that this protein has key functions in many different biological processes. This is to be expected bearing in mind the contributions of cell walls to many different aspects of plant life. AtTHE1 has also received attention because of its apparently more specific function in CWI maintenance in response to CWD. Interestingly, amongst higher plants, the CrRLK1L gene family has fairly conserved numbers and expression domains for its members, suggesting that their function might also be conserved. In contrast, the number of WAK family members, which also seem to be able to detect cell wall impairment, is increased significantly in other plant species compared to Arabidopsis. However, WAKs and WAKLs continue to be involved in cell elongation and responses to biotic stress mechanisms in different plant species. Therefore, the reason for the increased number of genes and potential benefits of having them remains an open question. One possibility is that expression of several of these genes is restricted to specialized tissues and organs not found in other species.By monitoring the status of their cell walls, plants are able to adapt successfully to adverse environmental conditions. Sensing environmental stimuli at the wall triggers cellular responses responsible for successful adaptation to the surrounding environment. Progress has been made in Arabidopsis to identify the function of several CWI maintenance sensors and the processes they regulate. Nevertheless, the molecular mechanisms, interacting partners and regulatory pathways of most of the putative CWI sensors have still not been elucidated in detail. Knowledge regarding the mode of action of the CWI maintenance mechanism and its relevance in other plant species is in its infancy. Larger genomes and multiple homologous genes, likely with redundant function, will pose difficulties in identifying the key molecular components for CWI maintenance. Investigating both ends of the evolutionary tree, close species to Arabidopsis on one side and ferns on the other, should help us discover common molecular mechanisms involved in the response to CWD and CWI maintenance and more importantly identify opportunities to use the resulting knowledge to improve performance of food and bioenergy crop plants.In Arabidopsis, our knowledge regarding transcriptional regulation of primary cell wall metabolism has started to grow recently. This is exemplified by the ERF family of transcription factors, which regulate expression of CESA genes, thus controlling cellulose production. Regarding other metabolic processes required for primary cell wall formation, our understanding could be improved. However, comparing this knowledge to information regarding transcriptional regulation of the same metabolic processes in other species, highlights that the situation is there even worse, thus creating exciting research opportunities. In contrast, information, knowledge and understanding regarding transcriptional regulation of secondary cell wall metabolism in Arabidopsis and other plants is much more advance as exemplified by our knowledge regarding the networks of TFs from different families acting in a hierarchical manner to control secondary cell wall formation. For several Arabidopsis homologues the functions seem to be preserved in other species suggesting they could form leads to analyse the regulatory processes in these species in a knowledge-based manner.


Alpha-synuclein (aSyn) is a 14 kD protein encoded by the SNCA gene that is expressed in vertebrates and normally localizes to presynaptic terminals and the nucleus. aSyn forms pathological intracellular aggregates that typify a group of important neurodegenerative diseases called synucleinopathies. Previous work in human tissue and model systems indicates that some of these aggregates can be intranuclear, but the significance of aSyn aggregation within the nucleus is not clear. We used a mouse model that develops aggregated aSyn nuclear inclusions. Using aSyn preformed fibril injections in GFP-tagged aSyn transgenic mice, we were able to induce the formation of nuclear aSyn inclusions and study their properties in fixed tissue and in vivo using multiphoton microscopy. In addition, we analyzed human synucleinopathy patient tissue to better understand this pathology. Our data demonstrate that nuclear aSyn inclusions may form through the transmission of aSyn between neurons, and these intranuclear aggregates bear the hallmarks of cytoplasmic Lewy pathology. Neuronal nuclear aSyn inclusions can form rod-like structures that do not contain actin, excluding them from being previously described nuclear actin rods. Longitudinal, in vivo multiphoton imaging indicates that certain morphologies of neuronal nuclear aSyn inclusions predict cell death within 14 days. Human multiple system atrophy cases contain neurons and glia with similar nuclear inclusions, but we were unable to detect such inclusions in Lewy body dementia cases. This study suggests that the dysregulation of a nuclear aSyn function associated with nuclear inclusion formation could play a role in the forms of neurodegeneration associated with synucleinopathy. Alpha-synuclein (aSyn) is a small 14 kD protein encoded by the SNCA gene that is expressed in vertebrate neurons and localizes to presynaptic terminals and the nucleus ,3; howevaSyn functions at the presynaptic terminal, where it can interact with the SNARE complex and participate in neurotransmitter vesicle cycling ; howeverPathological aSyn nuclear inclusions have been most often described in MSA, but there are also reports of certain kinds of nuclear aggregates being present in PDD and LBD, suggesting a potentially more widespread involvement of nuclear aggregates in many synucleinopathies ,14,16. AAlthough there are reports of nuclear inclusions in multiple model systems, the focus of many of these studies has been on the associated cytoplasmic pathology. For instance, an early transgenic PD mouse model expressing human aSyn under control of the PDGF promoter reported dense nuclear inclusions; however, the characteristics of these inclusions were not extensively described . We haveIn this study we aimed to better understand the role of nuclear inclusions in disease pathology using a mouse model that develops nuclear inclusions and an analysis of synucleinopathy patient tissue. Using aSyn PFF injections in GFP-tagged aSyn (Syn-GFP) transgenic mice, we were able to induce the formation of nuclear aSyn inclusions and study their fixed tissue and in vivo properties. To examine the relevance of nuclear inclusions in human disease, MSA and LBD brain tissue was screened for four types of inclusions, NCIs, NNIs, GCIs, and GNIs, and compared to our mouse model.We used in vivo multiphoton imaging of layer 2/3 of mouse sensory-motor cortex via a “cranial window,” with approaches we have previously developed, to study the presence and properties of nuclear aSyn inclusions ,10,26,32Alpha-synuclein in a pathologic conformation is highly phosphorylated at serine-129, and this pSyn is an established marker of Lewy pathology in human tissue . We usedTo better understand the molecular properties of NNIs, we stained both Syn-GFP-positive and -negative inclusions in fixed tissue with an antibody to GFP to confirm that the endogenous Syn-GFP signal colocalized with the anti-GFP antibody staining as expected . All incPathological brain tissue from several neurodegenerative diseases has been shown to contain linear inclusions composed of cofilin and actin that have been called “actin rods.” Although these actin rods are found in the cytoplasm in most diseases where they are described, actin rods have been reported within the nucleus in Huntington’s disease . To testTo understand how NNIs could relate to neurodegeneration within neurons that develop them, we used in vivo multiphoton imaging to longitudinally follow cortical neurons bearing somatic inclusions with and without detectible nuclear inclusions. Interestingly, this analysis of Lewy inclusion-bearing neurons in vivo over time suggests that specifically shaped aggregates of aSyn could predict upcoming cell death. Our previous work in this model suggested that when looking at all neurons that develop somatic Syn-GFP Lewy inclusions, they have a half-life of ~6 months after inclusion formation ,26. In taSyn nuclear inclusions are a well-defined characteristic of MSA ,23, but We have confirmed the presence of nuclear inclusions composed of aggregated aSyn both in human MSA and mouse model brain tissue. In our Syn-GFP mouse line, linear, mobile, rod-shaped Syn-GFP nuclear aggregates form spontaneously in a small proportion of neurons even without the induction of Lewy pathology, but this has no obvious consequence in terms of cell viability that we can detect. Further work will be required to determine if these spontaneously forming intranuclear rods contribute to specific alterations in cellular function or not. In contrast, aSyn PFF-induced NNIs are correlated with imminent cell death. These NNIs are in a subset of cells also containing cytoplasmic Lewy inclusions, induced to form by aSyn PFF injection. These NNIs in PFF-injected brain are not composed of actin, making them unlikely to be actin rods, but are composed of aSyn aggregated in an amyloid conformation and phosphorylated at serine-129, two hallmarks of Lewy pathology. In our study we did not test the proteinase K sensitivity of the intranuclear aSyn inclusions detected, neither in the mouse model nor MSA patient tissue. It will be informative in future work to determine whether these intranuclear inclusions are resistant to proteinase K, as has been described for somatic aSyn inclusions in MSA , and wheOne of the more intriguing observations from our data in PFF-injected mouse brain transgenically expressing Syn-GFP in a small subset of cortical neurons is the rare presence of NNIs composed of Syn-GFP in a neuron containing a cytoplasmic inclusion that is Syn-GFP negative. One possible explanation for this observation is that a trans-cellular transport of aSyn, potentially in an aggregated form, can occur between neurons and this involves the movement of aSyn out of one cell and into the cytoplasm and then nucleus of another cell. Although there is ample evidence that aSyn can be secreted and taken up by neurons in culture, and a leading model of PFF-induced Lewy pathology formation and spread in mouse brain suggests the trans-synaptic transport of a small aggregated aSyn “seed” species between neurons, to our knowledge, there is no previous data suggesting that aSyn internalized by a cell is delivered to its nucleus. There is elegant work, however, demonstrating that certain homeobox transcription factors can be secreted by cells and contain activity that allows them to translocate across the neuronal plasma membrane in a receptor-independent manner . These hA second, unexpected observation from our data is that the presence of NNIs with specific morphologies can predict upcoming neuronal death within a period of 14 days. Our previous work showed that neurons bearing cytoplasmic Lewy inclusions in this model have a half-life of 6 months or more, suggesting that neurons can live with these cytoplasmic inclusions for relatively long periods of time ,26. HoweMSA has long been described as a neurodegenerative disorder with nuclear inclusions. Papp and Lantos suggested that the formation of NNIs occurred early, before NCI formation, and even before aSyn was known to be the primary component making up these inclusions ,46. LateOur data suggest that aSyn nuclear inclusions are present in PFF mouse models of synucleinopathy and that they may play important roles in disease pathobiology. These nuclear inclusions are composed of serine-129 phosphorylated and aggregated aSyn in a fibrillar amyloid form that can potentially be transmitted between cells. Our in vivo multiphoton imaging data demonstrate that the presence of these nuclear inclusions in neurons predicts their impending cell death and suggest that the dysregulation of a nuclear aSyn function could play a role in this neurodegeneration. Further work should focus on better characterizing the molecular nature of these intranuclear inclusions, the processes that lead to their formation, and testing whether they are causally related to cell death. A deeper understanding of the mechanisms of nuclear aSyn aggregation and its consequences for brain cells could define new targets for therapeutic intervention in several important neurodegenerative diseases.Animals were housed by OHSU’s Department of Comparative Medicine in a light–dark cycle, temperature and humidity-controlled vivarium, and maintained under an ad libitum food and water diet. All experiments were approved by the OHSU IACUC. All experiments were performed in accordance with the relevant guidelines and regulations, and every effort was made to minimize the number of animals used and their distress. Syn-GFP animals have been previously described (PDNG78 ).Two- to three-month-old mice were injected with mouse wildtype sequence PFFs . Injections were performed according to published protocols for PFF generation, preparation, and cortical injections ,10. BrieImmunohistochemistry was carried out similarly to previously published protocols ,33. FixeElectron microscopy (EM) was carried out similarly to previously published protocols . AnimalsCranial window surgery, imaging, and analysis were conducted using a similar protocol as we have previously published for isoflurane-anesthetized animals at 4–5 months of age (2 months after PFF injection) ,26,33. WHuman subjects were seen in the Oregon Alzheimer’s Disease Center (OADC) and affected subjects had an established clinical diagnosis. Brain tissue from autopsies of ten patients diagnosed with Lewy body dementia and ten patients with multiple system atrophy was procured in a de-identified manner from the OADC neuropathology core. Tissue use was approved by the IRB at OHSU.Ten MSA and ten LBD cases were provided through the OADC neuropathology core. Standard histological methods were used to evaluate aSyn expression in the frontal gyrus, hippocampus, amygdala, and midbrain for all cases, and in the putamen and pons in MSA cases. Briefly, formalin-fixed, paraffin-embedded (FFPE) tissue sections were incubated with Syn1  or EP1536Y , developed with diaminobenzidine (DAB) chromagen, and counterstained with hematoxylin. Additionally, MSA cases were immunofluorescently labeled, and 7 μm FFPE sections from the pons and putamen were blocked in 5% BSA in PBS + 1% TritonX-100 (PBST) for 1 hr at room temperature before incubation overnight at 4 °C in EP1536Y  or Syn1  diluted in 1% BSA PBST. Appropriate secondary antibodies  and DAPI diluted in 1% BSA PBST were incubated for 1 hr at room temperature. Lipofuscin autofluorescence was quenched in 1× TrueBlack Plus . Immunohistochemical and immunofluorescent slides were imaged on a Zeiss ApoTome2 microscope and Zeiss LSM 980 confocal microscope, respectively.