Feline Immunodeficiency virus (FIV), similar to its human analog human immunodeficiency virus (HIV), enters the central nervous system (CNS) soon after infection and establishes a protected viral reservoir. The ensuing inflammation and damage give rise to varying degrees of cognitive decline collectively known as HIV-associated neurocognitive disorders (HAND). Because of the similarities to HIV infection and disease, FIV has provided a useful model for both in vitro and in vivo studies of CNS infection, inflammation and pathology. This mini review summarizes insights gained from studies of early infection, immune cell trafficking, inflammation and the mechanisms of neuropathogenesis. Advances in our understanding of these processes have contributed to the development of therapeutic interventions designed to protect neurons and regulate inflammatory activity. FIV, similar to HIV, is a lentivirus that infects and replicates in T cells and cells of monocyte lineage including perivascular macrophages and microglia ,9,10,11.FIV was isolated from naturally infected domestic cats soon after the identification of HIV  and was In contrast to HIV-1, FIV uses CD134 as a primary receptor instead of CD4 ,57. SimiFIV and HIV are thought to gain initial access to the CNS via trafficking of infected monocytes although other mechanisms may contribute such as trafficking of T cells, penetration of free virus across a damaged blood–brain barrier and trafficking across the blood-cerebrospinal fluid (CSF) barrier. Once the virus gains access to the CNS, infection spreads to microglia and astrocytes but not neurons. Virus production in the CNS is typically low and may be controlled in part by CD8+ T cells ,67,68,69One of the questions of interest has been whether virus in the CNS represents uniquely adapted quasispecies that may account for both neurotropism and neurovirulence. Studies of FIV envelope diversity during the early stages of infection have shown that the virus that first appears in the CSF is similar to plasma . Over tiGL8 [In vitro studies using fetal feline brain microvascular endothelial cells grown on transwell insert membranes containing 5 µm pores have been used to investigate trafficking of immune cells across the feline blood-brain barrier . These sGL8 . FIV proGL8 ,88. FIV GL8 ,89 even In addition to penetration across the blood-brain barrier, virus may also penetrate into the brain via the blood-CSF barrier. This barrier differs from the blood–brain barrier as there are no tight junctions at the vascular endothelium. This allows easier penetration of virus and immune cells into the choroid plexus stroma which contains a large population of macrophages and dendritic cells, excellent targets for infection by FIV. The blood-CSF barrier is formed by tight junctions between the cuboidal epithelial cells of the choroid plexus . Traffic5 macrophages/200 µL into the lateral ventricle) induced a massive influx of neutrophils throughout the brain in the absence of local infusion damage or bleeding. The effect was subsequently confirmed in rats using macrophages tagged with fluorescent polystyrene 1 µm spheres. The macrophages were tracked to the cervical lymph nodes but were not seen in the brain parenchyma. This observation, although limited, is an indication of the profound effect ventricular macrophages can have on immune cell trafficking into the brain. It is also a caution that the ventricular environment is an immunologically active environment. This should be taken into consideration in experiments employing intracranial infusions. Important questions remain concerning the sentinel functions of ventricular macrophages and their specific role in the regulation of the CNS immune environment.Interactions between macrophages and T cells could be particularly prominent within perivascular regions and within the choroid plexus where infected macrophages are commonly seen in HIV, simian immunodeficiency virus (SIV) and FIV infections. In addition, many studies have documented an admixture of systemic and CNS lentiviral transcripts within the choroid plexus of infected hosts ,100,101 Because the course of FIV-induced disease, including CNS infection, parallels HIV infection, FIV has been used as a model to investigate the processes that lead to neural damage. One of the difficulties associated with the use of FIV as a model for HIV-associated neurodegenerative disease is the very gradual decline in CNS function with few overt signs until after about 5–8 years, often overlapping with the development of acquired immune deficiency syndrome (AIDS). In addition, as in HIV-infected humans, only a small subset of infected cats show readily apparent neurological signs as they become immune deficient. A question often raised is whether cats naturally infected with FIV have neurological disease since there is little indication of clinically relevant neurological deficits. The brains of random source FIV infected cats examined post mortem show clear signs of degeneration. Less is known about well maintained domestic cats and several factors complicate the assessment of these cats. The deficits seen in HIV infected patients are cognitive-motor processing deficits with no primary motor deficits. In patients maintained on antiretroviral therapy cognitive decline reflects frontal cortex deficits such as attention and executive functions ,15. ThesIn contrast to the clinical setting, neural and behavioral dysfunction are commonly seen in experimentally infected cats using sensitive physiological and behavioral methods. Because these methods are complex and/or expensive they are not practical in the clinic. In addition, because of the slow course and low natural prevalence of neural deficits, experimental studies of FIV-associated neurological disease have adopted strategies to accelerate neuropathogenesis. These strategies have employed neonatal inoculation with FIV , the useIn studies of experimentally infected specific pathogen free cats, designed to mimic HIV infection in humans, neurological signs can be detected as early as 12 months post infection. The signs include abnormal, stereotypic motor behaviors, anisocoria, increased aggression, increased cortical slow wave activity in quantitative electroencephalograms, prolonged latencies in brainstem evoked potentials, delayed righting and pupillary reflexes, decreased nerve conduction velocities, changes in sleep architecture and deficits in cognitive-motor functions ,109,110.NCSU1) [The CNS pathology that develops in cats experimentally infected with FIV reflects the low grade inflammation seen in pre-AIDS/encephalitis patients and current patients on antiretroviral therapy. Findings include infiltration of perivascular macrophages, microglial activation, astrogliosis, myelin pallor, diffusely distributed signs of neural damage in the form of beaded and/or fragmented dendrites ,115,116 NCSU1) . The losWith the exception of advanced stages of disease progression where neuron loss becomes evident, the pathology largely reflects ongoing inflammation which is potentially reversible. An important observation that deserves greater attention is the development of Tau pathology in aged cats ,121,122.The availability of fetal tissue from cats provides the opportunity to perform in vitro experiments that explore the cellular processes that lead to pathology. Neurons are easily cultured from fetuses at approximately 35–40 days gestation (~6–7 cm fetus) ,129,130.In early studies of virus interactions with primary feline mixed neuronal cultures e.g., A, the adThe identity of the soluble factors that impair neuronal function is still largely unknown. While a diverse range of HIV-associated, macrophage-derived factors have been proposed to be responsible for neurotoxicity ,136,137,When conditioned medium from FIV-treated choroid plexus macrophages was added to cultured feline neurons (1:5 dilution) an acute increase in intracellular calcium was seen B followeOverall, these studies illustrate that inflammation sensitizes neurons in a fashion that encourages the development of more severe neurodegenerative pathologies. Therapeutic approaches that prevent the early neuronal dysfunction have the potential to interrupt the pathological cascade at a point where it is largely reversible. The FIV model offers many benefits for these efforts. Nevertheless, it is also important to recognize limitations of the model. Lack of species-specific reagents and assays can prevent detailed exploration of toxic factors and mechanisms. In spite of these limitations, the FIV model offers the ability to assess potential neuroprotective strategies both in vitro and in vivo.Insights from the above studies have supported a model of neuropathogenesis illustrated in NTR) has strong neuroprotective efficacy. At 10 nM, the ligand protected neurons from the long-term toxic effects of FIV in mixed neural cultures, suppressed the delayed accumulation of intracellular calcium (but not the acute increase) and greatly decreased dendritic damage [Neurotrophin ligands may act at receptors on neurons or macrophages/microglia to prevent the calcium dysregulation and cytoskeletal disruption. Because FIV neuropathology and neurodegenerative mechanisms are highly similar to HIV it provides the opportunity to test potential therapies in FIV infected cats. Studies of cultured feline neurons have shown that a novel neurotrophin ligand which targets the p75 neurotrophin receptor  the natural progression of CNS disease; (2) the role of astrocytes and microglia in immune cell trafficking across the blood-brain and blood-CSF barriers; (3) the mechanisms of early infection, viral diversification and disease progression in the CNS; (4) the potential importance of the choroid plexus blood-CSF barrier as a site of virus entry; (5) the potential mechanisms that lead to the loss of neuronal calcium homeostasis and neuronal pathology; and (6) have facilitated the identification and testing of novel treatments. The FIV model recapitulates many features of inflammation-associated neurodegeneration that are thought to promote various neurodegenerative diseases. The ability to investigate the pathogenesis of FIV in specific-pathogen free cats in parallel with in vitro studies of cultured feline cells provides the opportunity for translational studies that should facilitate the design and evaluation of new therapeutic strategies that prevent neurodegeneration.


Down syndrome (DS) is a genetic disorder caused by the presence of a third copy of chromosome 21. DS affects multiple organs, but it invariably results in altered brain development and diverse degrees of intellectual disability. A large body of evidence has shown that synaptic deficits and memory impairment are largely determined by altered GABAergic signaling in trisomic mouse models of DS. These alterations arise during brain development while extending into adulthood, and include genesis of GABAergic neurons, variation of the inhibitory drive and modifications in the control of neural-network excitability. Accordingly, different pharmacological interventions targeting GABAergic signaling have proven promising preclinical approaches to rescue cognitive impairment in DS mouse models. In this review, we will discuss recent data regarding the complex scenario of GABAergic dysfunctions in the trisomic brain of DS mice and patients, and we will evaluate the state of current clinical research targeting GABAergic signaling in individuals with DS. Down syndrome (DS) or trisomy 21 is the leading cause of genetically-defined intellectual disability and congenital birth defects. DS is characterized by many phenotypical features affecting almost all body systems, including developmental defects and growth delay Nadel, . In partAlthough cognitive impairment is the most common and severe feature of DS, other neurological and psychiatric manifestations of the disease highly impinge on the quality of life of individuals with DS and their families. In particular, the DS population shows increased frequency of anxiety .GABA is the main inhibitory neurotransmitter in the adult and healthy brain and it acts through ionotropic and metabotropic receptors , which maintains low intracellular chloride concentration ([Cl−]i). Thus, GABAAR opening generates an influx of negative chloride ions that hyperpolarizes the cell membrane potential and inhibits neuronal activity. Moreover, opening of GABAAR ion channels may also shunt concurrent excitatory currents , thus preventing them from bringing the membrane potential to the action potential threshold. Indeed, GABAAR opening short-circuits depolarizing synaptic currents by locally reducing the input resistance , which generates high [Cl−]i. In this condition, the chloride flux through GABAARs is outward and depolarizes the cell membrane potential  will be in most cases not sufficient to reach the threshold for action-potential generation, they will be able to activate voltage-gated calcium channels (VGCCs), remove the voltage-dependent magnesium block from NMDA receptors  GABAAR response may also occur in mature neurons depending on timing and location of GABAARs, upon relatively small difference in [Cl−]i at different cell compartments, more hyperpolarized resting membrane potentials, sustained activity or even pathological conditions  sits very near to the resting membrane potential (VREST), relatively small changes in [Cl−]i are sufficient to change the polarity of GABAergic responses. In particular, if ECl is more negative than VREST, the chloride flow will be inward (hyperpolarizing). Instead, if ECl is less negative than VREST, the chloride flow will be in the opposite direction, generating depolarizing PSPs.GABABen-Ari, . Thus, aARs are composed of five subunits, and the many subunit variants identified in mammals  confer different pharmacological and kinetic properties to the receptor  currents  mostly localized extra-synaptically. Their high affinity for GABA ensures that ambient GABA spilled out of synapses can activate GABABRs despite of their distance. In particular, the GABABR Gαi/o subunit can lead to adenylate cyclase inhibition and consequent reduction of cAMP levels and PKA pathway activity , tetramers formed by different compositions of GIRK1–4 subunits  currents in early neurodevelopment, although maintaining the pre-synaptic inhibitory function on neurotransmitter release. Indeed, coupling of postsynaptic GABABRs to Kir channels is delayed during development  and induction and monocular deprivation, respectively; Levelt and Hübener, ARs usually exert a negative regulation on hippocampal plasticity and cognition. Indeed, GABAAR-mediated inhibition suppresses LTP both in vitro and in vivo  or GABAAR activation impairs memory  of the hippocampus  and adult mice  databaseThe vast majority of the studies on DS-related cognitive and electrophysiological abnormalities have been performed on the Ts65Dn mouse. Indeed, although the Ts65Dn model still presents issues from a genetic point of view  individually trisomic (through a tandem duplication on the corresponding chromosome) for the three complete syntenic regions on Mmu10, 16 and 17, respectively  were found larger in the CA1 region of 2 weeks old Ts65Dn mice  to hyperpolarizing (inhibitory) during neuronal maturation and it can shift between inhibition and excitation under some circumstances and/or in particular cellular compartments  rescued the observed increase in GABAB-triggered currents in CA1 pyramidal neurons , maps to Hsa21  of synaptic efficiency following specific stimulation protocols. In particular, LTP represents the cellular correlate of memory and it is necessary for different cognitive processes, including learning Lynch, . In lineAR antagonist picrotoxin (PTX). Similarly, potentiation at perforant path-DGGC synapses (DG-LTP) was restored by application of either PTX or the GABABR antagonist CGP55845 . The effect was evident for up to 1–3 months after treatment cessation  was rescued in Ts65Dn slices by acute application in the recording bath of the GABAFinally, comparative evaluation of synaptic plasticity deficits in the other DS mouse models has shown that DG-LTP was similarly impaired and rescued by PTX application in Ts1Cje, Ts1Rhr, and triple trisomic (Dp10/Dp16/Dp17) mice  at non-epileptic doses on long-term declarative memory in the novel object recognition test in Ts65Dn mice: chronic (but not acute) blockade of GABAA transmission rescued object recognition memory after 2-weeks of treatment  on learning and memory in trisomic mice. We found that chronic NKCC1 inhibition in Ts65Dn mice was able to rescue discriminative memory in the object recognition test, spatial memory in the object location test and associative memory in the contextual fear conditioning task  cells in the somatosensory, visual and auditory cortices , a derivative of RO4938581, previously shown to rescue learning and memory in Ts65Dn mice  brain deficits originate during the embryonic and early life. Since activation of both GABA− accumulation during specific developmental periods, when brain circuits are possibly more prone to plastic changes, may result in beneficial effects in learning and memory that persist into adulthood. In this regard, it would be interesting to test the long-term effects of GABAAR inhibition  or of lowering [Cl−]i with Bumetanide during brain development. The importance of an early intervention during a likely essential period of brain development in DS is highlighted by the observation that different treatments  administered during gestation or in the early postnatal period rescued memory performances in Ts65Dn mice later in life  that affect the quality of their lives and may in turn also impinge on their cognitive abilities  induced epileptiform activity in Ts65Dn mice that was rescued by genetically restoring GIRK2 gene dosage to disomy  has been repeatedly described as a key regulator of sleep and circadian rhythms , a low predictive potential of the Ts65Dn mouse model may also have played a role. For example, changes in GABAAR subunit composition specific for human DS  may play a pathological role  during development and lead to complex synaptic, physiological and circuit changes, ultimately causing cognitive deficits and other neurological manifestations. As a consequence, the still unmet need of identifying effective pharmacological interventions to alleviate DS-related cognitive impairment represents an incredible complex challenge for the future. Surely, the insights about GABA-related impairments in DS models may be of great relevance also for other neurodevelopmental disorders where defective GABAergic transmission  and Telethon Foundation .AC and LC are named as co-inventors on International Patent Application PCT/EP2014/078561, filed on December 18, 2014, and connected US, EP, JP National Phase Applications, claiming priority to US Provisional Application US 61/919,195, priority date December 20, 2013. The other author SM declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


MYC as well as BCL2 and/or BCL6, has recently been formally categorized as “High grade B-cell lymphoma [HGBL], with rearrangements of MYC and BCL2 and/or BCL6” as per the 2016 revision of the World Health Organization (WHO) classification of lymphoid neoplasms. Patients with DHL can frequently present with adverse prognostic findings such as advanced clinical stage, elevated lactate dehydrogenase level and increased Ki67 expression on immunohistochemical staining [Double hit lymphoma (DHL), commonly defined as an aggressive B cell non-Hodgkin lymphoma (NHL) harboring rearrangement of staining .Initially-reported case series of DHL revealed poor clinical outcomes for patients with this disease regardless of therapy received , 5. A suMYC rearranged or MYC protein overexpressing aggressive lymphomas, including those with DHL, as either monotherapy or in combination with immunochemotherapy. These include the dual PI3 kinase/histone deacetylase inhibitor CUDC-907 (NCT02674750), the aurora kinase inhibitor alisertib (NCT02700022) and the immunomodulatory agent lenalidomide (NCT02213913). While use of these agents has led to disease response in preclinical models, their efficacy in the MYC-“altered” lymphoma, and specifically DHL, patient population remains to be seen.Given the overall poor prognosis of DHL, multiple novel agents are currently under investigation specifically for patients with MYC, such as the Arf-p53 tumor suppressor pathway or that of mitochondrial-induced apoptosis regulated by the Bcl-2 family of proteins [Based on variation in clinical presentation, as well as response to both standard and intensive front-line immunochemotherapy regimens experienced by patients diagnosed with DHL, it is apparent that biologic variation exists even within this subset of aggressive B cell NHL. This is supported by at least one study in which sequencing of a panel of genes frequently mutated in Burkitt lymphoma performed on tissue specimens from patients with DHL did not identify a common mutation pattern . It is pproteins , which mMYC, BCL2 and BCL6 in the evaluation of aggressive B cell NHL tissue specimens has led to increased recognition of cases of DHL, and consequently, appreciation of the varied clinical outcomes experienced by these patients . For this disease defined by a set of genetic abnormalities, it now seems prudent to search for additional molecular alterations that may inform prognosis, and more importantly, allow for the rational use of targeted therapies in an effort to increase in clinical benefit and minimize unwarranted toxicity in patients with DHL.More widespread utilization of cytogenetic testing to detect rearrangements in


Lactobacillus was the most abundant genus , followed by Lactococcus, Enterococcus, Vibrio, Weissella, Pediococcus, Enterobacter, Salinivibrio, Acinetobacter, Macrococcus, Kluyvera and Clostridium; this result was confirmed by q-PCR. PCoA based on Weighted UniFrac distances showed an apparent clustering pattern for Yucha samples from different locations, and Lactobacillus sakei, Lactobacillus saniviri and Staphylococcus sciuri represented OTUs according to the major identified markers. At the microbial functional level, it was observed that there was an enrichment of metabolic functional features, including amino acid and carbohydrate metabolism, which implied that the microbial metabolism in the Yucha samples tended to be vigorous. Accordingly, we further investigated the correlation between the predominant microbes and metabolic functional features. Thirteen species of Lactobacillus (147 strains) were isolated, and Lactobacillus plantarum (60 isolates) and Lactobacillus pentosus (34 isolates) were isolated from every sample.Yucha is a typical traditional fermented food of the Li population in the Hainan province of China, and it is made up of cooked rice and fresh fish. In the present study, metagenomic approach and culture-dependent technology were applied to describe the diversity of microbiota and identify beneficial microbes in the Yucha. At the genus level,  The Li population is an indigenous settlement of the Hainan province, which is located in the southernmost region of China. Fermented foods have played an important role in the Li daily diet since ancient times, which has been attributed to their rich taste and nutritionLactobacillus and 152 yeast strains in Airag and Tarag, and the Lactobacillus helveticus and Lactobacillus kefiranofaciens were the predominant bacterial speciesLactobacillus, Leuconostoc, Lysinibacillus and Bacillus as the predominant genera in these Zambian fermented samplesLactobacillus delbrueckii subspecies bulgaricus and Lactobacillus plantarum exhibited extreme polymorphisms associated with geographical distribution6Traditional fermented foods such as Yucha in the Hainan province of China are common around the world, and most studies have focused on the diversity of microbiota and the isolation of beneficial microbes in themThe Hainan province is located in tropical southern China, and it is separated from mainland China by the sea. The microbial resources are abundant and diverse in Hainan province because of its typical island-associated geographical featuresThe results of the microbial alpha diversity and acidity measurements are shown in Lactobacillus (Firmicutes phylum) was the most abundant genus , and the amounts of Lactococcus, Enterococcus, Vibrio, Weissella, Pediococcus, Enterobacter, Salinivibrio, Acinetobacter, Macrococcus, Kluyvera and Clostridium all exceeded 1%  among Yucha samples collected from the Qiongzhong, Baisha, Baoting and Changjiang areas. As shown in the kernel density figure , the UniLactobacillus sakei, Lactobacillus saniviri, Corynebacterium variabile and Staphylococcus sciuri represented OTUs  were enriched in samples from the Qingzhong area, though the amount of Lactobacillus acidophilus and Lactobacillus rossiae represented OTUs (OTU 57 and 153) was significantly lower than that found in samples from other areas. Meanwhile, the Pseudomonas, Lactobacillus acidophilus, Bifidobacterium, Subdoligranulum and Lactobacillus rossiae represented OTUs  were enriched in samples from Changjiang area as well as the Vibrio harveyi, Clostridium, Lactococcus piscium and Bacteroides plebeius represented OTUs  were enriched in samples from Baoting and Baisha areas.After confirming that there was a potential difference in the microbiota composition of the Yucha samples from different areas, we further identified differences in specific bacteria at the genus or species level. The OTU table was normalized according to the OTUs’ abundance across samples, and the significantly differences OTUs among samples in different sampling locations were clustered as the heatmap to give a better pattern across samples . It can To better understand the important role of the microbiota present in Yucha samples, PICRUStLactococcus, Acinetobacter and Kluyvera and all metabolic functional features. Meanwhile, Vibrio and Weissella were negative correlated with other functional features. Additionally, a significant positive correlation was found between Lactobacillus, which was the most abundant genus in the Yucha samples, and the metabolism of carbohydrates and nucleotides. Meanwhile, we calculated the contribution rate of the predominant genera to functional features related to microbial metabolism  were isolated as pure cultures from the Yucha samples. Among these Lactobacillus isolates, eighty-nine strains were isolated from MRS agar, and fifty-eight strains were isolated from M17 agar. Molecular identification results and the phylogenetic tree based on 16S rRNA gene sequencing are shown in Lactobacillus plantarum (60 isolates) and Lactobacillus pentosus (34 isolates) isolated from every sample were the predominant Lactobacillus species found in Yucha samples. Moreover, Lactobacillus farciminis (16 isolates), Lactobacillus brevis (10 isolates), Lactobacillus rhamnosus (6 isolates), Lactobacillus casei (6 isolates), Lactobacillus coryniformis (5 isolates), Lactobacillus senioris (2 isolates), Lactobacillus namurensis (2 isolates), Lactobacillus fermentum (2 isolates), Lactobacillus buchneri (2 isolates), Lactobacillus rossiae (1 isolate) and Lactobacillus crustorum (1 isolate) were isolated from Yucha samples collected from the settlements of the Li populations in the Hainan province of China.  One hundred forty-seven Lactobacillus accounted for 43.82% of the total number of sequences; thus, this genus was the predominant one found in the Yucha samples; this result was also confirmed by q-PCR. Furthermore, thirteen species of Lactobacillus were isolated by a culture dependent method. Generally, lactic acid bacteria are a group of Gram-positive bacteria that are able to ferment glucose and lactose to lactic acid and are beneficial to human healthLactobacillus is a symbiotic bacterium found in fish intestinal microbiota, where it plays a key role in maintaining the balance in the gut micro-ecological environment12Lactobacillus found in our samples was likely rooted in the raw fish and the surrounding environment. In addition, the Lactobacillus proliferated and produced lactic acid, thereby creating an acid environment that inhibited the growth of other bacteria.In the present study, a combination of next-generation sequencing and culture-dependent technology was applied to investigate the microbial community of Yucha. It could be observed that the reads of At the functional level, a general positive correlation between metabolic functional features  and core microbiota in the Yucha samples was highlighted. Fermented foods are a significant part of the daily diet of many people both in the Li populations and around the world. During the production of a fermented product, microorganisms transform raw material into a product with an increased value, generally by extending the shelf life of the raw materials and increasing the nutritional value of the product by improving the production of organoleptic attributes14Leuconostoc, Lactobacillus and Weissella were the predominant genera in Kimchi, and a genetic profile characteristic of heterotrophic lactic acid fermentation of carbohydrates was revealed in the Kimchi microbiome. Compared with the microbial diversity between Kimchi and Yucha samples, the genera Lactobacillus and Weissella were predominant bacteria. However, the most abundant genus Leuconostoc in Kimchi was not detected in present research. The Leuconostoc was a common genus of lactic acid bacteria, and its abundant enzyme systems encoded by various genomic islands for fiber and polysaccharides fermentation were revealed by previous researchLeuconostoc was often isolated in vegetal fermented foods. The Yucha is made of cooked rice and fish , so the micro-ecological environment was not suitable for the growth of Leuconostoc. In functional level, Jung focused on the microbial metabolic pathway of carbohydrate categories during Kimchi fermentation. And in Yucha samples, besides of the carbohydrate metabolism, the amino acid metabolism  of the microbes present in Yucha may render them suitable for further exploration and appropriate applications by the scientific community.In this study, 24 Yucha samples were aseptically collected from different Li settlement families  in the Hainan province of China. Samples were stored at 4 °C during the transfer to the laboratory and were analyzed within a few hours. The pH of the samples was measured, and the Total Titratable Acidity (TTA) values were determined according to previously described methodsTen grams of Yucha sample was homogenized with 90 mL sterile NaCl solution  to a homogenous suspension for 10 mins. DNA was extracted from the suspended material by using a QIAGEN DNA Mini-Kit  in combination with a bead-beating methodThe V3-V4 region of the 16S ribosomal RNA (rRNA) genes was amplified for each sample. A set of 6-nucleotide barcodes was added  to the u22Lactobacillus, Lactococcus, Acinetobacter, Weissella, Enterococcus and Salinivibrio as target microbes for quantification. The primers used for detecting the genera listed above and the reaction mixture (20 μl) were prepared as reported previouslyThe predominant bacteria of the Yucha samples were detected by quantitative PCR. Quantitative PCR was performed in an ABI Step-One detection system (Applied Biosystems). Based on the microbial abundance detected by the high-throughput sequencing, we chose the genera of Lactobacillus. Characteristic colonies were picked from each plate for morphology analysis. Isolates from the MRS and M17 agar were examined for a Gram reaction and catalase production. Cell morphology and motility were examined via the microscopic observation of cells grown in broth for 24 h (Olympus BX 40). DNA was extracted from Lactobacillus isolates according to Andrighetto et al.Ten grams of Yucha was homogenized with 90 mL sterile NaCl solution  to a homogenous suspension, and a tenfold serial dilution was carried out. Then, 100 μl of each dilution was spread onto the surface of MRS  and M17  agar and incubated at 37 °C for two days to isolate The genomic DNA of each isolation was used as a template for PCR amplification, and the 16S rRNA gene was amplified by primers 27F (5′-AGAGTTTGATCCTGG CTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′). The PCR procedure was performed as reported previouslyThe sequences were visualized and manually aligned using the DNAMAN software (v5.0). Sequence homology was examined by comparing the obtained sequences with those in the NCBI database. The phylogenetic trees were constructed using the neighbour-joining distance method in the 6.0 version of the MEGA program. The confidence values of branches of the phylogenetic tree were determined using bootstrap analyses based on 500 random resamplings.The raw sequences were trimmed to remove the low-quality sequences. The sequences were filtered according to quality scores using the sliding window approach, in which sequences are trimmed when the average quality score over a 50-nt sliding window drops below 30. More than 85.3% sequence retained after filtering. Bioinformatic analyses were performed using QIIME (v1.6)https://www.r-project.org/). Based on the rarefied OTU subset, the relative abundances of taxa were compared by Kruskal-Wallis test. False discovery rate (FDR) values were estimated using the Benjamini-Yekutieli method to control for multiple testingAll statistical analyses were conducted with the R programme .How to cite this article: Zhang, J. et al. Metagenomic approach reveals microbial diversity and predictive microbial metabolic pathways in Yucha, a traditional Li fermented food. Sci. Rep.6, 32524; doi: 10.1038/srep32524 (2016).


P < 0.05) and females . Indices of autonomic control of BP and HR were obtained by measuring BP and HR response to ganglionic blockade, β-adrenergic, and muscarinic receptors antagonists. HFD increased vascular but reduced cardiac sympathetic drive in males . HFD did not alter females' vascular or cardiac sympathetic drive. HFD specifically reduced aortic α-adrenergic constriction in males and lowered HR response to muscarinic receptor antagonism in females. These data suggest that obesity-associated increases in HR could be caused by a reduced cardiac vagal tone in females, while HR increases in males may compensate for the reduced vascular adrenergic contractility to preserve baseline BP. These data suggest that obesity impairs autonomic control of cardiovascular function in males and females, via sex-specific mechanisms and independent of fat distribution, metabolic disorder or inflammation.Obesity-related cardiovascular disease (CVD) involves increased sympathetic activity in men and male animals. Although women exhibit increased visceral fat, metabolic disorders, inflammation and CVD with obesity, whether body weight gain affects autonomic control of cardiovascular function in females remain unknown. Due to the lack of adequate model to mimic the human pathology, this study aimed to develop a murine model, which would allow studying the sex-specificity of the response of the autonomic nervous system to obesity and identifying the origin of potential sex-differences. We tested the hypothesis that sexual dimorphisms in the autonomic response to obesity disappear in mice matched for changes in body weight, metabolic and inflammatory disorders. Male and female C57Bl/6 mice were submitted to control (CD) or high fat diet (HFD) for 24 weeks. Female mice gained more adipose mass and lost more lean mass than males but reached similar visceral adipose mass and body weight, as males, at the end of the diet. 24 weeks of HFD matched male and female mice for visceral adiposity, glycaemia, plasma insulin, lipids, and inflammatory cytokines levels, demonstrating the suitability of the model to study human pathology. HFD did not elevate BP, but similarly increased heart rate (HR) in males (CD: 571 ± 9 vs. HFD: 631 ± 14 bpm,  The worldwide epidemic of obesity is a leading risk factor for cardiovascular disease (CVD) and a major health burden, in men and women. Due to the protective effects of female sex hormones on the cardiovascular system , it was determined that 21 weeks of HFD are required for male and female mice to develop a similar level of obesity. Mice were monitored for 24 weeks. Body weight was measured twice weekly. Food intake was determined in a sub-group of animals placed in metabolic cages for 3 days, 21 weeks after the initiation of the HFD treatment. Mice were housed in an American Association of Laboratory Animal Care–approved animal care facility at Augusta University.All surgical procedures and experimental protocols were approved by the Institutional Animal Care and Use Committee of Augusta University, in accordance with the guide for the care and use of laboratory animals published by National Institute of Health  Committee for the Update of the Guide for the Care and Use of Laboratory Animals, 2011). Male and female C57Bl/6 mice  were divided in 4 groups and fed either a control diet  or a high-fat diet  After 21 weeks of control or HFD, body composition was analyzed by nuclear magnetic resonance spectroscopy [EchoMRI (magnetic resonance imaging)]. Transmitters were implanted as described previously , of the β-adrenergic receptor blocker propranolol (6 mg/kg) and of the muscarinic receptor blocker atropine (1 mg/kg) in conscious mice  and euthanized via decapitation, in accordance with our approved animal protocol. Trunk blood and organs were respectively collected for plasma isolation and weight measurement. Fasting blood glucose and HbA1c levels were measured using a glucometer  and A1CNow  respectively. Plasma total cholesterol and triglycerides were measured with enzymatic colorimetric assays . Plasma insulin and leptin levels were determined using ELISA kits from ALPCO Diagnostics  , respectively.Thoracic aortas were dissected surgically, cleaned of fat, cut in 2 mm-long rings and mounted on a wire myograph  with 1 g of basal tension . For all comparisons, P < 0.05 was considered statistically significant.All data are expressed as means ± SEM. Data were analyzed using a two-way analysis of variance (ANOVA) followed by a Bonferroni At 6 weeks of age male and female mice had comparable body weight Table . In respCirculating levels of the adipocyte-derived hormone leptin were similarly increased in male and female mice on the HFD Table . GlycemiAs inflammation is a common feature of obesity we measured plasma levels of two cytokines associated with the progression of CVD, IL-6, and TNF-α  similarly increased body weight in male and female mice and abolished the sexual dimorphism in metabolic and inflammatory responses to obesity observed with short-term HFD treatments, (2) did not elevate blood pressure but increased heart rate to a similar extent in male and female mice, and (3) revealed a sex-specific response of the autonomic nervous system to obesity reflected by an enhanced vascular neurogenic support of blood pressure in males, a lower cardiac sympathetic tone in males and a reduced cardiac vagal tone in female mice. Relevant to these observations is the mouse model of obesity, the dimorphic response of the autonomic nervous system to obesity and the demonstration that, in agreement with our initial hypothesis, similar levels of adiposity, metabolic and inflammatory disorders impair autonomic control of the cardiovascular system in male and female mice.Compelling evidence from the literature indicates that induction of obesity with a relative short HFD treatment in rodents induces a sexual dimorphism in body weight, fat distribution, metabolic alterations, and degree of inflammation. Indeed, after 8–16 weeks of HFD, female mice are generally leaner and do habitually exhibit reduced increases in body weight, preserved metabolic function and lower degree of inflammation as compared to their male counterparts , a R01 from the NHLBI (1R01HL130301-01 to EB) and Start-up funds from Augusta University.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. The reviewer JC and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


Eubrontes-like and Grallator-like ichnogenera. The Mafube morphotypes are tentatively attributable to large and small tridactyl theropod trackmakers, possibly to Dracovenator and Coelophysis based on the following criteria: (a) lack of manus impressions indicative of obligate bipeds; (b) long, slender-digits that are asymmetrical and taper; (c) often end in a claw impression or point; and (d) the tracks that are longer than broad. To enable high-resolution preservation, curation and subsequent remote studying of the morphological variations of and the secondary features in the tracks, low viscosity silicone rubber was used to generate casts of the Mafube tracks.Footprint morphology  is one of the key diagnostic features used in the interpretation of ancient vertebrate tracks. Over 80 tridactyl tracks, confined to the same bedding surface in the Lower Jurassic Elliot Formation at Mafube , show large shape variability over the length of the study site. These morphological differences are considered here to be mainly due to variations in the substrate rheology as opposed to differences in the trackmaker’s foot anatomy, foot kinematics or recent weathering of the bedding surface. The sedimentary structures  preserved in association with and within some of the Mafube tracks suggest that the imprints were produced essentially contemporaneous and are true dinosaur tracks rather than undertracks or erosional remnants. They are therefore valuable not only for the interpretation of the ancient environment  but also for taxonomic assessments as some of them closely resemble the original anatomy of the trackmaker’s foot. The tracks are grouped, based on size, into two morphotypes that can be identified as  The fluvio-lacustrine and aeolian rocks of the Upper Triassic - Lower Jurassic Elliot and Clarens formations (Karoo Supergroup) preserve not only a range of vertebrate body fossils, but also a plethora of vertebrate invertebrate and plant ichnofossils that collectively record information about the dynamics of a ∼200 Ma old palaeoecological system in southern Africa . FurtherThe vertebrate footprints in the Upper Triassic to Lower Jurassic Elliot Formation have been assigned to multiple tetrapod genera some 45 years ago largely by The Mafube tracks are preserved as impressions (negative relief or concave epireliefs) of the original foot on the upper bedding plane of a very finegrained, horizontally laminated sandstone that is 40 m below the contact between the Elliot and Clarens formations . In addiGrallator-Anchisauripus-Eubrontes plexus  of Massospondylus sp together with crocodylians remains (Protosuchus haughtoni) and a jaw from a large, basal archosaurian reptile  from farms Glen Skye (121), Helpmekaar (251), La France (379), Saaihoek (194), Bloemhoek (330), Naudes Lust (334), Mizpah (164), De Villiersdrift (338), Langkloof (34), and Foutanie (331); (b) cynodonts (only Tritylodon sp.) from farms Saaihoek (194), Bloemhoek (330), Naudes Lust (334), and Langkloof (34); (c) basal archosaurian reptile  from farms Foutanie (331); and (d) amphibians (skull and jaw fragments) from farms De Villiersdrift (338) and Langkloof (34). It is noteworthy that a large partial femur of the “Highland Giant,” a herbivorous sauropod was discovered only <17 km ENE of Mafube at the same stratigraphic level as the Mafube track site in close proximity to the contact of the Elliot and Clarens formations , track #31 (Surface II) and track #57 (Surface III). For casting of the tracks, we used a platinum cure, two-part component silicone rubber . The choice to use room temperature vulcanization (RTV) silicone rubber over latex rubber was due to the low levels of shrinkage, high strength and flexibility, as well as the long shelf life of the former. What makes the RTV silicone rubber ideal for field use is its pot life of 50 min, a short curing time (4 hours at 25°C), and a mixing ratio of one to one of its components . Prior to casting, the four chosen tracks were carefully cleaned with a wet brush and then dried naturally. Individual tracks were surrounded by a retaining wall of non-curing modelling clay in order to prevent the silicone rubber from spilling beyond the foot impression . This, hThe Mafube tracks are preserved in a 300 m long, 2 m thick, very fine grained, yellow to cream-coloured, tabular sandstone unit within the uEF . InternaThe primary sedimentary structures in the sandstone unit are horizontal laminations, low-angle crossbedding  and 3D, Hexagonal casts of desiccation cracks that penetrate into the sandstone are preserved on the exposed track palaeosurface  and 3F aOver 80 tracks have been identified at the Mafube dinosaur track site  over a lV-shaped hypices with lengths constrained between ∼25–35 cm and widths of ∼20–30 cm. The morphology of the digit tips is somewhat obscured by the sandstone natural cast but appears to be predominantly V-shaped. Tracks #3, 6, 7 and 11  and small (#16 of morphotype B) tridactyl tracks as well as tracks of particular interest  interdigit angles of 35°.9–2.7 cm ; Table 1Track #68 lacks distinct morphology, however digits III and ‘ridges’ within digit II or IV (digit cannot be adequately identified) may be inferred . Therefore, at Mafube, the solidification of the imprinted layer had to be relatively rapid, because the deposition of the overlying sediment layer occurred without producing erosional features. Instead, during the deposition of the overlying sediment layer, sandy sediment infilled  the delicate desiccation cracks and depressions between ripple marks as well as theropod foot impressions. Given that the sedimentary structures in the overlying very fine grained, horizontally laminated sandstones indicate upper flow regime conditions, the pervasive solidification of the palaeosurface was likely enhanced not only by subaerial exposure , but potentially also by microbial mats that biostablised the granular, sand-rich sediment layer, and ultimately produced a more cohesive and thus less erodible substrate cf. ; Fig. 10Tracks found to date at the Mafube are tridactyl, barring several which are poorly preserved  giving the impression of being didactyl. The anatomical detail of the tracks decreases towards the southern end of the site. The lack of manus impressions is indicative of obligate bipeds and eliminates quadrupedal trackmakers. We link the Mafube tracks to theropods based on the following criteria that distinguishes tracks of tridactyl theropods from ornithischians: (1) long, slender digits, that are asymmetrical, taper; and (2) often end in a sharp claw impression or point  and an anomalously high abundance of specimens are known from the Upper Triassic Chinle Formation  ratio averages 1.0 for the six better preserved specimens.Grallator-like tracks are also digitigrade and tridactyl with narrow interdigit divarication angles and Grallator by Grallator occurs globally , Hartford, Deerfield and Newark basins of USA by The size of morphotype B (length ∼15 cm) is comparable to those tracks that were described as Eubrontes, Grallator impressions are narrower with a length to width ratio ≥2 and the outer digits diverge at an angle of 10–30° despite the current ichnotaxonomic debate. Several other ichnotaxa are considered either behavioural/preservational variations  or junior synonyms of the four commonly considered valid ichnogenera above. Size has long been the key separating criteria in the GrallatorAnchisauripusEubrontes or GAE allometric plexus  has been long considered and debated  to be more evocative of Eubrontes and the smaller morphotype B to be more similar to Grallator.The foregoing also highlights the plight of assigning relatively large tridactyl tracks to the le e.g., . For exaSedimentological, taphonomic and ichnological assessments of the >80 tridactyl theropod tracks at Mafube dinosaur track site:  1.Reveal casts of desiccation cracks preserved in impressions of true dinosaur tracks which are valuable for reliable taxonomic assessments; 2.Demonstrate how track shape variability is linked to changes in the substrate consistency along the same palaeosurface ; 3.Confirm animal movement towards a water source despite the multi-directionality of tracks and lack of trackways on the palaeosurface; 4.Confirm the earlier reconstructions of the Lower Jurassic uEF depositional environment and reinforce the interpretation of a fluvio-lacustrine setting, typified by high energy, ephemeral water courses with flash flood-type discharge under semi-arid climatic conditions; 5.Eubrontes and Grallator ichnogenera.Aid the Lower Jurassic ecological assessments by providing evidence that during the deposition of the uEF theropod dinosaurs of variable size were present and left behind tracks possibly representing the 10.7717/peerj.2285/supp-1Table S1Additional anatomical measurements of Mafube tracks relating to the size differential between morphotypes A and B at the Mafube dinosaur track site. See Click here for additional data file.


Although larger species of animals typically live longer than smaller species, the relationship of body size to longevity within a species is generally opposite. The longevity advantage of smaller individuals can be considerable and is best documented in laboratory mice and in domestic dogs. Importantly, it appears to apply broadly, including humans. It is not known whether theses associations represent causal links between various developmental and physiological mechanisms affecting growth and/or aging. However, variations in growth hormone (GH) signaling are likely involved because GH is a key stimulator of somatic growth, and apparently also exerts various “pro-aging” effects. Mechanisms linking GH, somatic growth, adult body size, aging, and lifespan likely involve target of rapamycin (TOR), particularly one of its signaling complexes, mTORC1, as well as various adjustments in mitochondrial function, energy metabolism, thermogenesis, inflammation, and insulin signaling. Somatic growth, aging, and longevity are also influenced by a variety of hormonal and nutritional signals, and much work will be needed to answer the question of why smaller individuals may be likely to live longer. In this context, it is interesting to consider to what extent somatic growth and adult body size can influence the trajectory of aging and life expectancy. Pioneering studies of Samaras and his colleagues provided numerous examples of negative association of human height and lifespan.Deciphering relationships between growth and longevity in human populations is difficult because both can be powerfully influenced by environmental factors including nutrition, numerous public health measures, access to medical interventions and lifestyle factors such as smoking, alcohol, and drug use. In this article, we will briefly review evidence for the links between somatic growth, aging, and longevity in experimental animals and discuss the most likely underlying mechanisms.Caenorhabditis elegans) or in a fly (Drosophila melanogaster)17 are an important part of the present understanding of the genetic control of aging. The striking effects of mutations of a single gene on longevity of heterothermic invertebrates led to the question of whether identifiable “longevity genes” also influence aging and lifespan in mammals, including our own species. Studies conducted during the last 25 years in laboratory stocks of house mice (Mus musculus) demonstrated that longevity of these animals can be extended by a natural loss-of-function mutation or targeted deletion of a single gene.27 With very few exceptions, the genetic modifications which produced a significant and reproducible extension of longevity in both females and males disrupted the so called “somatotropic axis,” that is, biosynthesis or action of the pituitary growth hormone (GH) and/or the insulin-like growth factor I (IGF-1), an important mediator of GH actions. Since in mammals the somatotropic axis is a key regulator of postnatal growth, these long-lived mutants exhibit major reductions in growth rate and in adult body size. It should be emphasized that the remarkably extended longevity of GH-deficient and GH-resistant mice is associated with multiple signs of delayed aging and with the extension of “healthspan,” the period of life free of functional deficits and chronic disease.28 This includes maintenance of normal cognitive function (learning and memory) into advanced age.30 The rate of aging of these long-lived mutants was initially reported to be either reduced or unaltered, with life extension being due to a delay, rather than a slowing, of age-related mortality.31 However, a more recent analysis using what we believe is a more pertinent methodology suggests that the rate of aging of long-lived, GH-deficient and GH-resistant mice is initially slower than in genetically normal (control) animals and accelerates only later in their life, after most of the control animals have died.32The very exciting and largely unexpected discoveries of mutations that significantly extend longevity in a worm  aging.35The reciprocal relationship of longevity to growth rate and adult body size discovered in various dwarf mutants also applies to animals in which somatic growth and adult body size are experimentally enhanced. Transgenic mice, chronically expressing heterologous (in most cases bovine or human) GH in liver and other organs under control of metallothionein I or phosphoenolpyruvate carboxykinase promoters, grow faster than normal animals and achieve a greater adult body size, often exhibiting a very striking giant phenotype.37 and, more recently, in comparisons of individual animals from a normal, genetically heterogeneous population.38Experimental animals with extreme phenotypes, such as encountered in genetic dwarfs and giant transgenics, are useful for identifying the underlying mechanisms and previously unsuspected physiological relationships. However, it is of obvious importance to determine whether the associations and causal relationships described in these animals apply to organisms that have not been genetically altered and to genetic and phenotypic variations within the normal range. In fact, the negative association of longevity with adult body weight has been demonstrated in comparisons of normal (“wild type”) animals form different stocks, inbred strains and selected lines in studies going back to the ‘60 s40 It is interesting, but currently difficult to explain, why these relationships within species are opposite to the fairly consistent trend for large species of mammals and birds living longer than smaller species. One could speculate that because smaller species are more vulnerable to predation and other environmental hazards, they have developed life-course strategies for early reproduction and high fecundity. This, in turn, may divert available resources away from repair and maintenance and thus lead to a shorter lifespan. The surprising tendency of circulating IGF-1 levels to be lower, rather than higher, in large species41 may also prove to be a contributing factor to their longevity.Importantly, the negative association of body size and longevity extends to other mammalian species, with differences in longevity between different breeds of domestic dogs, and between individual dogs differing in size providing the most striking example.21 Defining the role of these mechanisms in the extension of healthspan and lifespan in the examined mutants is complicated by the fact that many of the differences between mutant and control (“wild type”) animals could represent either mechanisms or markers of delayed and/or slower aging. For example, long-lived Ames dwarf and growth hormone receptor knockout (GHRKO) mice are more insulin sensitive than their normal siblings42 but insulin sensitivity generally declines during aging.44 Thus, this difference could simply confirm the fact that at the same chronological age, the long-lived dwarf mice are biologically younger. This difficult issue was addressed in one of our studies by comparing gene expression data in long-lived mutants to two groups of normal animals that were either of the same chronological age or a comparable “biological age,” that is their age represented a similar percent of their life expectancy.45 In this particular study, differences in hepatic expression of the examined genes in GHRKO vs. normal mice were shown to be due to the differences in genotype rather than in biological age.45 However, we are not aware of a similar analysis being performed in other long-lived mutants or in studies examining other candidate mechanisms of aging.The (very consistent) negative association of adult body size and longevity in laboratory mice and other mammalian species brings up the questions of causality and underlying mechanisms. Available data can be interpreted as evidence that normal growth involves some intrinsic “costs” in terms of aging and longevity. Thus, faster or longer growth, and the consequent attainment of greater body size, somehow predispose the organism to earlier and/or faster aging and a shorter lifespan. The underlying mechanisms could be envisaged to influence growth with a secondary impact on aging and lifespan, or to independently influence both growth and aging, possibly via different signaling pathways or cellular processes Fig. . In eith21 Mechanisms most likely related to the negative association of somatic growth and longevity and to the opposite effects of GH signaling on these processes are briefly discussed below.Detailed discussion of all the mechanisms that appear to be involved in linking reduced somatotropic signaling to slower aging and extended longevity is outside the scope of this review. Interested readers are referred to several recent review articles.46 While activation of mTOR signaling prevents cell death, promotes protein synthesis, growth and cell divisions, it apparently also accelerates aging. Blagosklonny and his colleagues suggested that aging can be viewed as “a continuation of developmental growth driven by genetic pathways such as mTOR”.47 These investigators also proposed that gender differences in longevity of mice may be related to greater mTOR activity in males48 and that conversion of reversible cell cycle arrest to senescence (termed “geroconversion”) represents one of the mechanisms by which mTOR promotes aging.49 The excess “developmental” growth driven by mTOR presumably contributes to the accumulation of unfolded proteins, endoplasmic reticulum (ER) stress, and reactive oxygen species (ROS) production, and leads to inhibition of autophagy and promotion of cell senescence.The ability of GH to directly or indirectly activate the mTOR signaling pathway provides one of the most likely explanations of how this hormone can exert positive effects on somatic growth and negative effects on the lifespan. Mechanistic TOR (mTOR) pathway integrates somatotropic, nutritional and stress signals and plays a role in the control of autophagy and cell senescence.52 mTOR exerts its function via two complexes: mTORC1 and mTORC2. There is increasing evidence that the “anti-aging” effect of mTOR suppression is due to inhibition of mTORC1 signaling. For example, long-lived mice with disrupted somatotropic axis have diminished TORC1 signaling, while TORC2 activity may be increased55 mTORC2 appears to act to prevent, rather than promote aging, and consequently, its inhibition can reduce longevity. The evidence for anti-aging effects of mTORC2 signaling includes demonstration that genetic deletion of Rictor, a mediator of TORC2 signaling, reduces longevity in male mice.56 Interestingly, body weight and accumulation of white adipose tissue (WAT) in these animals were increased, suggesting that mTORC2, in contrast to mTORC1, may normally act as a negative regulator of growth.57 Further support of the role of TORC-2 in the control of aging, adipose-specific deletion of Rictor leads to increases in body size, weight of non-adipose organs and levels of IGF-1, IGFBP3, and insulin with a concomitant decrease of adiponectin, i.e., produces phenotypic characteristics generally associated with accelerated aging.57Pharmacological or genetic suppression of mTOR signaling pathways results in extended longevity in organisms ranging from yeast to mice58–60 The long-lived diminutive GHRKO and hypopituitary dwarf mice, in fact, exhibit increases in the mass and activity of brown adipose tissue (BAT), the key site of non-shivering thermogenesis, along with evidence for thermogenic activation, the so-called “browning” of WAT .63 Reduced RQ implies increased reliance on fatty acids as a metabolic fuel which is generally considered a marker of improved mitochondrial efficiency leading to generation of smaller amounts of noxious ROS.67 In fact, reduced RQ in long-lived dwarf mice is associated with increased expression of genes involved in β-oxidation of free fatty acids  and with reduced ROS generation.68 These alterations, together with improved anti-oxidant defenses69 probably account for less oxidative damage to DNA and other macromolecules in these long-lived animals.71 Although it remains to be conclusively proven which of these associations are causally related to aging, we believe that improvements in mitochondrial function in response to increased demand for thermogenesis represent one of the mechanisms linking reductions in somatotropic signaling, growth and adult body size to slower (and/or delayed) aging and extended longevity.73Another mechanism which may be contributing to the longevity benefits of small body size involves adjustments in mitochondrial function and energy metabolism in response to greater heat loss and increased demand for thermogenesis. Smaller individuals have a greater body surface to body mass ratio and, thus, lose heat faster. Therefore, they have to increase thermogenesis to maintain body temperature. This physiological response is of particular significance in laboratory mice whose thermoneutral temperature is ~30 °C (86 °F) and, thus, housing at a “standard” animal room temperature  represents a chronic mild cold stress.42 promoting cell senescence74 and chronic low grade inflammation,76 as well as favoring differentiation and depletion of at least one type of stem cell.77 Thus, the spectrum of known biological actions of GH could explain why growth and adult body size would tend to be negatively associated with longevity.78In addition to the mechanisms discussed above, the negative association of body size and longevity within a species likely reflects several indirect, but important, mechanisms. These include pleiotropic actions of GH which affect both body size and aging, acting through different, but likely physiologically related mechanisms. Growth hormone is a key regulator of hepatic IGF-1 expression, circulating IGF-1 levels, growth and adult body size, but it also appears to promote aging by a variety of actions such as inducing insulin resistance81 Severe GH resistance in the syndrome of Laron dwarfism produces a remarkable degree of protection from cancer.82 Neoplastic disease may have little if any influence on aging, but it has an obvious and major impact on longevity. This is particularly striking in laboratory mice in which cancer is an important and, in many strains, by far the most common cause of death. The fact that GH induces insulin resistance, an important element of the metabolic syndrome, prediabetes and diabetes, should also be mentioned in this context. The risk of diabetes is increased in association with abnormally elevated GH levels in acromegaly83 and reduced in the GH-resistant individuals with Laron syndrome.84Another indirect link between body size and longevity concerns alterations in disease risk. Growth hormone and IGF-1, as well as stature, have been related to cancer incidence on the basis of numerous in vitro, in vivo and epidemiological studies.86 Rapid growth in response to food availability after a period of nutrient shortage, the so-called “catch-up growth”, may be particularly important in this context.89 Increased risk of chronic disease in adult life can obviously reduce longevity and it also may represent a symptom of accelerated aging.91 Results of recent and ongoing studies in our laboratory indicate that treatment of hypopituitary Ames dwarf mice with GH injections during a relatively brief period (6 weeks) during development increases adult body size and significantly reduces the remarkably long lifespan of these animals.93 Importance of early  GH actions in the control of longevity is indirectly supported by the recent report that disruption of the GH receptor gene in adult mice produces a relatively modest increase in lifespan only in females.94Impacts of nutrition on growth, body composition, disease risk and aging are outside of the scope of this brief article, but can provide yet another indirect mechanism for the observed associations. Thus, both pre-natal and post-natal overnutrition can stimulate growth, as well as obesity and also increase risk of various chronic diseases in adulthood.19 Chronic thyroxine treatment can shorten longevity of Snell dwarf mice,95 as well as normal rats 96 while subclinical hypothyroidism was associated with exceptional longevity in women.98 However, treatment of dwarf mice with thyroxine limited to the peripubertal period did not alter their longevity.99It should also be mentioned that the endocrine basis of the complex relationships between growth-related processes and aging is not limited to the somatotropic axis. Ames and Snell dwarfs, which represent some of the extremes of mouse longevity, are both GH- and thyroid stimulating hormone-deficient and, thus, profoundly hypothyroid.100 However, chronic elevation of glucocorticoid levels can promote age-related pathology, and is suspected of contributing to accelerated aging of GH transgenic mice.101The relationships between the activity of the hypothalamic-pituitary-adrenal axis and aging are complex and poorly understood.Much more work will be needed to determine whether the association of reduced body size with extended longevity is truly causal, and to identify the mechanisms underlying this association.Negative association of longevity with GH signaling, somatic growth and adult body size discovered in GH-deficient and GH-resistant mutant mice applies also to genetically normal individuals and to other mammalian species. Stimulation of mTORC1 singling by the somatotropic axis could explain this association but many other mechanisms appear to be involved.


Fanconi anemia is an inherited disease characterized by genomic instability, hypersensitivity to DNA cross-linking agents, bone marrow failure, short stature, skeletal abnormalities, and a high relative risk of myeloid leukemia and epithelial malignancies. The 21 Fanconi anemia genes encode proteins involved in multiple nuclear biochemical pathways that effect DNA interstrand crosslink repair. In the past, bone marrow failure was attributed solely to the failure of stem cells to repair DNA. Recently, non-canonical functions of many of the Fanconi anemia proteins have been described, including modulating responses to oxidative stress, viral infection, and inflammation as well as facilitating mitophagic responses and enhancing signals that promote stem cell function and survival. Some of these functions take place in non-nuclear sites and do not depend on the DNA damage response functions of the proteins. Dysfunctions of the canonical and non-canonical pathways that drive stem cell exhaustion and neoplastic clonal selection are reviewed, and the potential therapeutic importance of fully investigating the scope and interdependences of the canonical and non-canonical pathways is emphasized. Not all patients have a suitable donor, and successful transplants are not infrequently associated with the subsequent development of solid tumors and other unique ‘late effects’17, so less toxic forms of therapy are clearly desirable. Their development does, however, require a more complete understanding of molecular pathogenesis than any of us has today. The first hurdle cleared on this point of pathogenesis was the identification of the commonly mutated genes. Since the cloning of the first gene (FANCC) in 199219, 20 other genes have been identified  is a rare inherited syndrome characterized by progressive bone marrow failure (BMF) and a high relative risk of hematopoietic and epithelial malignancies in children who sometimes have characteristic developmental abnormalities, including short stature and radial ray defects. Since the first clinical report of this disease 90 years agontified  and otheFANCB located on chromosome X25 and autosomal dominant mutations of FANCR26). For a DNA sequence to be considered an FA gene, an inactivating mutation must be associated with classic chromosomal breakage in response toex vivo challenge with cross-linking agents (mitomycin C or diepoxybutane) in at least one patient and complementation with the wild-type version of the mutant alleles must correct the aberrant chromosomal breakage response. Most patients will have signs of BMF and a high likelihood of developing epithelial malignancies early in life, but few patients  develop malignancies without first showing clinical signs of marrow failure27. Whether the hematopoietic stem cells (HSCs) ofFANCM patients are truly ‘fit’ is still in question because HSCs in theFANCM-deficient murine model have not been stringently testedin vivo 28.FA is an autosomal recessive disorder (except those rare cases associated with mutations of54 for monoubiquitinylation of two other proteins (FANCD2 and FANCI)57. This post-translational modification is required for the ‘downstream’ activation of other proteins that unhook ICLs (FANCP and FANCQ) and facilitate homologous recombination 58. Other nuclear functions of FA proteins in mitosis prevent aneuploidy and aberrant centrosome accumulation61. This review focuses on molecular pathogenesis in FA hematopoietic tissues and addresses the question of whether the canonical functions of the FA proteins account for all of the phenotypic features characteristic of this disease.Every FA gene encodes a protein that functions unambiguously to facilitate repair of cross-linked DNA. In response to interstrand crosslinks (ICLs), a core complex of eight proteins  is required62 is conclusive proof that the repopulating capacity and overall fitness of the mutant HSCs are unambiguously reduced during development and can be overtaken by even one HSC in which the mutation has been corrected. This conclusion has been confirmed in human embryonic stem cells63 andFancc–/– andFancd2–/– mice65. The experiment of nature62 provides clear theoretical evidence that if a single corrected stem cell can so massively outperform all of the mutant ones during development, gene therapy for this disease early in life (or perhapsin utero66) can be successful in the future. In fact, many preclinical and a few clinical gene therapy studies have been completed or are ongoing72. Although there have been no clear successes in patients yet73, the goal is within sight and entirely rational.Although clinical marrow failure progresses over time in the lives of patients with FA, it is clear that HSCs are defective early in development. That functionally corrective somatic mutations in a single human HSC prenatally are capable of completely replacing the hematopoietic tissues in twins by the time of birthsine qua non of FA diagnosis, until recently it had not been clear that cross-linking agents, apart from those that might be lurking in the external environment, played a role in the BMF so commonly found in FA. Work by Patel and colleagues has provided some important answers on this point. Having observed that alcohol dehydrogenase-5 (ADH5) deficiency results in synthetic lethality when combined with FA gene suppression in the chicken DT40 cell line74, the group confirmed that loss of either ADH575 or aldehyde dehydrogenase-2 (ALDH2)76—enzymes that catabolically inactivate formaldehyde and acetaldehyde, respectively—markedly increases the intensity and onset of severe BMF inFancd2-deficient mice. Given the universal intolerance of FA cells to cross-linking agents (the basis of the gold-standard diagnostic test), it is not surprising that reducing the catalysis of endogenous cross-linking agents would make the disease worse, but what is clear from these studies is that endogenous aldehydes represent major threats to FA-HSCs. Based upon these findings, it was soon discovered that Japanese FA patients with the dominant negativeALDH2 allele (rs671) have accelerated BMF77. Some investigators have proposed that steady-state endogenous aldehydes themselves account for marrow failure through DNA damage-induced cell death78. This might be true but more experiments need to be done on this score because aldehydes are capable of cross-linking many more types of molecules than just DNA79 and some cross-linked non-DNA targets stimulate two other inter-related processes known to be involved in FA pathogenesis, namely inflammation and oxidative stress82. Autophagosome formation can also be directly inhibited by singlet oxygen-mediated cross-linking of p62, a scaffold protein normally involved in proper autophagosome formation and function83. Moreover, inflammatory processes can suppress the expression of ALDHs in other non-hematopoietic tissues as well as macrophages87. Therefore, in seeking pharmacological remedies for BMF in FA, one needs to consider not only agents that activate or otherwise enhance the function of ALDHs88 but also the basic mechanisms leading to their suppression in the face of inflammation and other stressful cues. It should also be considered that normal FA proteins might enhance the activity of ALDH isoforms directly or indirectly.Although hypersensitivity to cross-linking agents remains theThe coordinated interactions of FA proteins play an essential role in responding to environmental toxins, and the loss of function of any one of them destabilizes genomes. Because these proteins are so unambiguously essential in nuclear processes that protect and repair the genome, there has been a widespread tendency to simply attribute HSC dysfunction to accrued DNA damage and cell death resulting from the lost canonical functions of the proteins in DNA repair. However, recently, studies have shown that at least some of the FA proteins participate in control of signaling pathways that stress HSCs and the loss of those functions can play a role in disease progression.100, some of which are independent of other FA proteins or effected outside the nuclear envelope93 and some are functionally independent of the DNA damage response104, and (2) important endogenous factors that suppress FA HSC function are emerging and include proteins normally involved in the inflammatory response108, pathways functionally linked to or activated by inflammation, including oxidative stress117, mitophagy102, and the production of endogenous aldehydes118. Some of the non-canonical functions of FA proteins are listed in120, excessive apoptosis when challenged with inflammatory cytokines or redox stress122, and ultimately progressive stem cell attrition in the face of inflammatory stress123.Two things have become clear: (1) while all FA proteins play a role in stabilizing the genome, many of them have additional functions126, earlyin vitro studies on FA cells were conducted to test the idea that FA cells might somehow be hypersensitive to this and other known inhibitory cytokines . These studies were positive127 and led to the identification of some non-canonical signaling pathways with which FA proteins were directly involved93. These early studies were conductedin vitro, but more recent work has unambiguously confirmed that murine FA stem cells are uniquely vulnerable to inflammatory stresses of many different kinds123 and that, in one informative model, blocking the function of the inflammatory cytokine interleukin 1 beta (IL-1β) (a suppressor of FA HSC proliferationin vitro)130 prevented marrow failure123. Multiple inflammatory cytokines and adhesion molecules are involved in the inflammatory response, including factors that can enhance and others that inhibit the replicative capacity of hematopoietic stem and progenitor cells (HSPCs), so it will be important to sort out whether the ‘replicative stress’ induced in the FA HSPC pool plays a role in damaging the cells or whether the suppressive and pro-apoptotic and differentiation-inducing effects of inflammatory cytokines cause them to perish. But the inflammatory response and its association with oxidative stress  provide opportunities for pharmacological intervention either in the ground state or during episodes of overt inflammation.Because of the known myelosuppressive role of interferon gamma (IFNγ) in the pathogenesis of acquired aplastic anemia107. Interestingly, mutations of FANCA, FANCG, and FANCC enhance the binding interaction of FANCC and PKR and result in PKR hyperactivation as well142. The recently described differential FANCA-binding functions of HSP90 and HSP70 and the functional consequences of such differential binding on the canonical function of various FANCA mutant substrates143 add to the complexity of the futurein vivo studies now required to confirm that these effects are truly independent of induced DNA damage. Finally, that ALDH and hsp70 are induced by and collaborate to control certain viral infections144 suggests that the relationships between ALDH, heat shock proteins, IFN-dependent pathways, and FA proteins should also be formally examined because they may be perturbed interdependently in FA cells.A variety of complex indirect effects likely account for some of the inflammatory vulnerabilities of FA HSCs. For example, wild-type FANCC interacts with hsp70 to suppress activation of eukaryotic translation initiation factor 2-alpha kinase 2 (PKR) in wild-type cells exposed to IFNγ and double-stranded RNA or IFN and TNF. The interaction involves the canonical substrate-binding domain of HSP70, and loss of the FANCC/hsp70 interaction results in hyperactivation of PKR in FA cells145. So the pharmacological suppression of induced cytokine production or function may also reduce the stress on the HSPC pool. Some preliminaryin vitro evidence of this had been reported145, but the scope of non-canonical signaling defects in FA is increasingly broad and involves interdependent pathways that pose a challenge for picking a particular molecular focus. Cross-talking environmental cues that induce inflammation, for example, regularly induce oxidative stress149 and enhance endogenous aldehyde loads86. Any one of these stressors can excessively suppress FA HSPCs and induce hyperactive inflammatory cytokine production in more well-differentiated myeloid cells.One of the additional challenges associated with the aberrant FA inflammatory response is that FA mononuclear phagocytes, the progeny of FA HSCs, overproduce such cytokines in response to Toll-like receptor (TLR) activation150), thereby clearing damaged mitochondria and reducing reactive oxygen species. Knockdown ofFANCC,-F, or -L also leads to defective mitophagy101. Importantly, the function of FANCC in Parkin-mediated mitophagy is independent of its role in genomic DNA damage repair102. This point was recently demonstrated by using aFANCC mutant which does not rectify the dysfunction induced by FA mutations in the canonical pathway but does correct the Parkin-mediated mitophagic function of the protein102. This sameFANCC mutant  also rescues defective STAT1 activation inFANCC-deficient cells without correcting the canonical pathway92 and is reportedly associated with a milder phenotype151, suggesting that parallel non-canonical pathways contribute to progression of BMF in FA.Some FA proteins now provide novel functional insights on long-recognized ties between inflammation and oxidative stress. Many of the non-canonical functions outlined in152, PKR91, p38 MAPK141, the TLR pathway (p38 MAPK and MK2)132, and, most recently, hyperactive transforming growth factor beta (TGFβ) signaling94. The ideal therapeutic agent would be one thatin vivo: (1) reduces chromosomal breakage of all FA somatic cells exposed to endogenous and exogenous cross-linking agents, (2) enhances survival of FA cells exposed to genotoxic agents, (3) relieves the defects in FA HSCs in the ground state and in the face of inflammatory challenges, (4) reduces overproduction of or aberrant responses to reactive oxygen species, (5) enhances the metabolic inactivation of endogenous aldehydes, and (6) relieves the overproduction of inflammatory cytokines in more well-differentiated cells (hematopoietic and otherwise). In fact, the TGFβ ‘pathway’ results in many of these favorable responses in FA cells94, so it is currently an appealing target.In small-molecule therapy of FA, target candidates could include hyperactive signaling pathways in mutant cells that include p53 and p21Fancd2–/– HSCs, increased the percentage of quiescent HSPCs inFancd2-deficient mice after exposure to polyinosinic:polycytidylic acid (pI:pC), and prevented pI:pC-induced marrow failure. Interestingly, the titer of DNA damage in mutant HSPCs was also reduced by the treatment with the antibody, a finding that led to the discovery that TGFβ inhibition enhanced homologous recombination repair of double-strand breaks (more likely to be error-free153) and decreased non-homologous end joining (error prone153) in FA cells and the confirmation that the same effect could be achieved by using a small-molecule inhibitor (SD208)94. Why the TGFβ pathway is hyperactivated in FA cells is unclear, so a number of important questions remain to be addressed, including the potential impact on induced cytokine production in FA auxiliary cells  and on expression of various ALDHs in both auxiliary cells and HSCs. The role of non-SMAD signaling pathways155 ought to be tested, particularly since some of them are known to be involved in TLR hyper-responsiveness in FA macrophages131 and in suppression of FA erythropoiesisin vitro145. Paracrine and autocrine sources of such factors must be identified and HSPC subtypes need to be carefully investigated because responses to TGFβ pathway disruption of various subsets can differ substantially156. Ruling out functional interference with other molecules of the 33-member TGFβ superfamily157 159 would help address safety concerns for any candidate molecule as would consideration of the anti-oncogenic function of TGFβ1 in modulating the transforming activity of HOXA9160. This latter point is essential given the known high risk in FA patients of clonal evolution to myelodysplastic syndrome or acute myeloid leukemia. Nonetheless, although more work is required, the identification of the TGFβ pathway as a potential therapeutic target is buttressed by having met more standards for pharmacological candidacy than any other small-molecule approach to date.Antibody-mediated inhibition of the myelosuppressive protein TGFβ enhanced survival and performance of murineparallel pathways that have evolved to protect the genome and protect germ cells162 and HSCs. This makes evolutionary sense. In multicellular organisms, this would simply ensure that loss of FA protein function would be attended by loss of fitness in the stem cell pool, limiting the rapid emergence of mutants. Unfortunately, in patients with FA, loss of HSC fitness leads most frequently to life-threatening BMF.Loss of the non-canonical functions of the FA proteins likely serves to exacerbate or activate processes that result in HSC dysfunction. Involvement of these proteins in protection from oxidative stress, inflammatory cues, and endogenous aldehydes and in facilitating selective autophagic responses occurs, at least in part, by way of165). In some cases in which mosaicism occurredin utero, the progeny of one mutant stem cell replaced the entire hematopoietic systems of twins, neither of whom ever developed BMF62. The second pathway, stem cell death and stem cell exhaustion, leads to the first of two undesirable outcomes, BMF. This is the most common early life-threatening complication in patients with FA. The third pathway is the selection of neoplastic stem cell clones. In this process, the loss of HSC fitness in the face of such an unstable genome creates an opportunity for selection of stem cell clones that have gained, throughsomatic mutations, a new capacity to either exploit or resist the very factors that had previously controlled their state of unfitness.In nature, three things can happen to FA HSCs and two of them are undesirable. HSCs take the one good path least commonly. Here, a single FA HSC can mutate in a way that causes reversion of the mutation on one of the FA alleles  phase23. This process has also been confirmed in FA mice. Specifically, exposure ofFancc-deficient HSPCs to both growth factors and the inhibitory factor TNFin vitro suppressed their growth compared with the growth of wild-type cells, but over time, in some cases, rapidin vitro growth of FA-deficient cells ensued. The rapidly proliferating cells had clonal cytogenetic defects and had acquired resistance to TNF. When transplanted into congenic wild-type mice, they induced fatal acute myelogenous leukemia166. The canonical pathway in these surviving leukemic cells was still fully defective but they had gained a survival advantage by ‘quasi-adaptively’ developing cytokine resistance.For example, FA hematopoietic progenitor cells are characteristically suppressed by TNF and IFNγare involved and contribute in a major way to the selection of neoplastic clonal hematopoiesis. Second, it is worth re-emphasizing that the murine models of FA develop truly fulminant BMF only when an inflammatory response is provokedin vivo123, so even if the non-canonical functions play a minor role, interdiction of the non-canonical phenomena that provoke stem cell attrition should have therapeutic value. In fact, Eklund’s group has shown that this is the case at least in FA mice123. These observations have created some enthusiasm for attacking the molecular basis of stress intolerances in FA cells. They also create hope that by reducing selective pressures and selective inflammatory sweeps through the FA HSC pool, BMF could be prevented or forestalled and neoplastic clones would not emerge.The idea of multifunctionality of some of the FA proteins has met with some resistance in large part because the non-canonical processes affected can be either the cause of DNA damage or a consequence of it. To those who would argue that non-canonical pathways play a minor role or no role at all in reducing fitness of FA HSCs and that all non-canonical functions of FA proteins described to date are simply downstream consequences of DNA damage, I would raise two points. First, the natural selection of ‘adapted’ stem cell clones serves as evidence that non-canonical pathways167.In summary, the advances in the FA field are exciting. They have resulted not only from a focus of the research community on the biochemistry of DNA damage repair but from an increasing recognition that the FA proteins are multifunctional and participate directly in biochemical pathways that effect protective responses to endogenous aldehydes, oxidative stress, inflammation, mitophagy, and virophagy. This increasingly complex expansion of the FA field may lead to more holistic clarity on molecular pathogenesis not only of BMF, leukemogenesis and cancer but of skeletal and cutaneous manifestations of this disease. Finally, today we recognize that this disease provides a uniquely informative window through which we can view more clearly the potential role of selective sweeps in the pathogenesis of myeloid neoplasms in the general population