Body fluid levels of neuroactive amino acids in autism spectrum disorders: a review of the literature

A review of studies on the body fluid levels of neuroactive amino acids, including glutamate, glutamine, taurine, gamma-aminobutyric acid (GABA), glycine, tryptophan, d-serine, and others, in autism spectrum disorders (ASD) is given. The results reported in the literature are generally inconclusive and contradictory, but there has been considerable variation among the previous studies in terms of factors such as age, gender, number of subjects, intelligence quotient, and psychoactive medication being taken. Future studies should include simultaneous analyses of a large number of amino acids [including d-serine and branched-chain amino acids (BCAAs)] and standardization of the factors mentioned above. It may also be appropriate to use saliva sampling to detect amino acids in ASD patients in the future—this is noninvasive testing that can be done easily more frequently than other sampling, thus providing more dynamic monitoring.

Autism spectrum disorders
The term autism spectrum disorders (ASD) refers to neurodevelopmental diseases that affect 1-2 % of children, according to the data on the broad array of ASD (Baron- Cohen et al. 2009). ASD is characterized by different levels of severity and occurs in all ethnic groups. Noto et al. (2014) reported that 1 out of 88 children aged 8 years will develop an ASD, with males more at risk than females. Blumberg et al. (2013) showed that the prevalence of ASD had risen 75 % from 2007 to 2012 in the United States. Feng et al. (2013) identified 12 studies when searching Chinese databases in 2013; the prevalence in these studies varied from 2.8 to 29.5/10,000.
ASD is characterized by impaired social interaction skills combined with restrictive/repetitive behaviors (American Psychiatric Association 2013). Genetic predisposition and environmental factors undoubtedly have effects on the pathophysiology of ASD, but the precise mechanisms related to the pathophysiology of ASD are unknown and definitive methods for prevention or treatment are lacking (Blaylock 2008). DSM-5 proposes that ASD symptoms must appear in the early childhood (infant) (American Psychiatric Association 2013). Receiving diagnosis at an early stage of development could contribute to the early intervention and therapy, benefiting both patients and their families Sacrey et al. 2015;Brian et al. 2015). However, behavioral abnormalities are often overlooked in the early stage of ASD, even experienced professionals involved in pediatric healthcare (Howlin and Asgharian 1999). Therefore, many researchers have been trying to establish quantitative diagnostic criteria that could contribute to an early and more accurate ASD diagnosis. Many interacting factors are probably contributing to the etiology of ASD, and these potential factors are described in several excellent review articles (Lam et al. 2006;Pardo and Eberhart 2007;Aoki et al. 2012;Parellada et al. 2014;Lozano et al. 2015;Rozas et al. 2015;Subramanian et al. 2015;Zhang et al. 2015;Martin et al. 2016;Muller et al. 2016;Park et al. 2016); this review focuses on amino acids.

Neuroactive amino acids
Several lines of evidence have shown that changes (e.g., availability, metabolism, and/or receptor activity) in neuroactive amino acids associated with central brain functions may play a role in the pathogenesis and/or pharmacotherapy of several psychiatric disorders (e.g., schizophrenia and mood disorders) that have symptoms, such as cognitive impairment and problems with social interactions, in common with ASD (Coyle 2006;Grant et al. 2006;Lam et al. 2006;Labrie et al. 2008;Ongür et al. 2008;Yüksel and Öngür 2010;Durrant and Heresco-Levy 2014). Several preclinical and clinical studies have implicated neuroactive amino acids in the etiology of ASD, fragile X syndrome, and tuberous sclerosis complex (TSC), but most of these studies have focused on glutamate, GABA, and/or glutamine (El-Ansary and Al-Ayadhi 2014; Rojas 2014; Santini et al. 2014;Rozas et al. 2015;Cochran et al. 2015;Lozano et al. 2015;Robertson et al. 2016). Other amino acids could also be involved and it may be important to conduct comprehensive studies in which a number of these amino acids are investigated simultaneously. Due to the potential role of neuroactive amino acids in the pathogenesis and treatment of ASD, monitoring changes in their concentrations in body fluids are also important in case they may be relevant to the early diagnosis and intervention in patients with ASD. This paper reviews the literature on such measurements of several of these important compounds in body fluids in ASD subjects.

Glutamate (GLU)
GLU, which is highly concentrated in brain, is the primary excitatory neurotransmitter (Naaijen et al. 2015). GLU normally has a protective effect with regard to neural plasticity and cognitive function, but excessive GLU may be neurotoxic, leading to death of neurons and glia (Olney 1969;Manev et al. 1989) and may possibly play a role in the pathogenesis of psychiatric disorders, such as ASD (Sheldon and Robinson 2007). Ghanizadeh (2011) and El-Ansary and Al-Ayadhi (2014) showed that GLU is involved in neuroinflammation in ASD, and Ghanizadeh and Namazi (2010) proposed GLU and homocysteine as targets for therapy of ASD patients' irritability and aggression. A hyperglutamatergic hypothesis of ASD has been proposed (Fatemi 2008;Blaylock and Strunecka 2009). Fatemi et al. (2002) showed that the levels of GAD 65 kDa and GAD 67 kDa proteins, both of which are involved in converting GLU to gamma-aminobutyric acid (GABA), are reduced in the brains of patients with ASD, resulting in the increased levels of GLU in the brain.
Studies on GLU levels in the plasma of patients with ASD compared to healthy controls (HCs) are contradictory, with some reporting increased levels (Moreno-Fuenmayor et al. 1996;Aldred et al. 2003;MacDermot et al. 2005;Shimmura et al. 2011;Tirouvanziam et al. 2011;Naushad et al. 2013;El-Ansary and Al-Ayadhi 2014;Cai et al. 2016) and some decreased levels (El-Ansary 2016). In addition, increased levels have been reported in serum (Shinohe et al. 2006) and decreased levels in platelets (Rolf et al. 1993) and urine samples (Evans et al. 2008;Yap et al. 2010;Nadal-Desbarats et al. 2014). Some studies have used magnetic resonance spectroscopy (MRS) to measure amino acid levels in patients with ASD and have reported that patients with ASD have increased GLU levels in brain (Page et al. 2006;Joshi et al. 2012;Hassan et al. 2013). Cochran et al. (2015) showed that, compared with HCs, patients with ASD had increased glutamine (GLN) levels, decreased GABA levels, and no difference in GLU levels in brain. However, van Elst et al. (2014) reported that GLU and GLN levels were decreased in ASD brains.

Glutamine (GLN)
GLU is stored in the form of GLN in astrocytes until it is transferred to presynaptic terminals and converted back to GLU (Magistretti and Pellerin 1999). Ghanizadeh (2010) reported that a glutamine (GLN) synthetase inhibitor may improve inflammation in ASD. Shimmura et al. (2011) suggested that the level of GLN in plasma could be a screening test for detecting ASD in children, especially those with a normal intelligence quotient (IQ). In studies of GLN levels in ASD patients compared to HCs, plasma (Moreno-Fuenmayor et al. 1996;Aldred et al. 2003;Shimmura et al. 2011;Tirouvanziam et al. 2011;Good 2011a;Tu et al. 2012; El-Ansary 2016) and platelet (Rolf et al. 1993) levels have been reported to be decreased, serum levels to be no different (Shinohe et al. 2006), and urine levels either increased (Noto et al. 2014) or decreased (Evans et al. 2008).

Taurine (TAUR)
TAUR is an osmoregulator and neuromodulator that suppresses vasopressin and has been reported to be depleted in urine of autistic children (Good 2011a). However, in other studies on TAUR levels in ASD patients compared to HCs, plasma levels have been reported to be increased (Moreno-Fuenmayor et al. 1996;Shimmura et al. 2011;Kuwabara et al. 2013) or decreased (Geier et al. 2009;Kern et al. 2011;Tu et al. 2012), and levels in urine samples to be increased (Yap et al. 2010;Nadal-Desbarats et al. 2014) or decreased (Ming et al. 2012). Although the results reported on TAUR levels in plasma and urine samples are contradictory, there is a consistent opinion that TAUR plays a protective role in patients with ASD (Good 2011a, b;Omura et al. 2015). Kuwabara et al. (2013) showed elevated plasma TAUR levels in adults with ASD and proposed that TAUR is compensatory against pathogenesis of ASD, such as that caused by oxidative stress.

Gamma-aminobutyric acid (GABA)
The balance between GABA and GLU, inhibitory and excitatory neurotransmitters, respectively, is very important for brain function, and many psychiatric and neurological disorders may be the result of imbalance between GABA and GLU (Erickson et al. 2013;Rojas 2014;Robertson et al. 2016). Reduced GABAergic action in human and animal models of ASD has been proposed to be one of the reasons for an imbalance between excitation and inhibition (Rubenstein and Merzenich 2003;Gogolla et al. 2009;Blatt and Fatemi 2011;Ito 2016;Robertson et al. 2016). However, compared with HCs, GABA levels in plasma have been reported to be increased in ASD subjects (Dhossche et al. 2002; El-Ansary and Al-Ayadhi 2014). Dhossche et al. (2002) reported that plasma GABA levels tended to decrease with age in ASD. Compared with HCs, GABA levels in platelets have been reported to be decreased in ASD (Rolf et al. 1993) while those in urine samples increased (Cohen 2002). Neuroimaging techniques reported decreased GABA in brains of ASD patients (Kubas et al. 2012;Gaetz et al. 2014;Rojas et al. 2014;Omura et al. 2015;Cochran et al. 2015). Rojas et al. (2014) reported that the left perisylvian GABA levels were decreased in patients with ASD and their unaffected siblings. Recently, studies using oxytocin to treat animal models of ASD reported that oxytocin can increase excitatory GABA and enhance hyperglutamatergic activity (Tyzio et al. 2014;Young and Barrett 2015).

Glycine (GLY)
GABA and GLY are major inhibitory neurotransmitters in the central nervous system (CNS). They act on receptors coupled to chloride channels which play an important role in normal function of the CNS (Ito 2016). GABA and GLY depolarize membrane potentials, acting as excitatory neurotransmitters during early development (Wang et al. 2002;Yamada et al. 2004;Kaila et al. 2014). They shift from excitatory to inhibitory neurotransmitters at birth and in maturation, and if that does not happen it may result in neurological disorders, including ASD (Tyzio et al. 2006(Tyzio et al. , 2014. However, in some parts of the brain, GLY acts as a coagonist at NMDA GLU receptors (Martina et al. 2003;Baptista and Varanda 2005;Kim et al. 2005;Basu et al. 2009), and it has been suggested that the GLY/d-serine site on the NMDA receptor could be a target for ASD therapy. Compared with HCs, GLY levels in plasma (Tirouvanziam et al. 2011) and serum (Shinohe et al. 2006) of ASD subjects have been reported to be unchanged, and levels in urine samples reported to be increased (Nadal-Desbarats et al. 2014;Noto et al. 2014) or decreased (Evans et al. 2008;Ming et al. 2012).

Tryptophan (TRP)
Serotonin (5-hydroxtryptamine, 5-HT) is an important neurotransmitter, and TRP is the precursor of serotonin (Zhang et al. 2015). Dysfunction of serotonin systems are implicated in some forms of ASD, and may contribute to social interaction impairments (Lam et al. 2006;Rubin et al. 2013;Yang et al. 2014). Whole blood serotonin has been reported to be elevated in at least 25 % of ASD children (Muller et al. 2016). However, reducing TRP in the diet can impair social behavior in patients (McDougle et al. 1996) and mice (Zhang et al. 2015) and increasing TRP in the diet has been reported to improve social behavior in mice (Zhang et al. 2015). Compared with HCs, TRP levels in plasma have been reported to be decreased in ASD (Tirouvanziam et al. 2011;Tu et al. 2012;Naushad et al. 2013), while Noto et al. (2014) reported levels to be increased in urine samples and Kałużna-Czaplińska et al. (2014) reported them to be decreased.

d-Serine
In the recent years, d-serine in the brain has been the subject of extensive research (Fuchs et al. 2005(Fuchs et al. , 2011Nunes et al. 2012;Billard 2015;Sacchi et al. 2016). d-Serine is an important amino acid in glutamatergic transmission (Fuchs et al. 2005) and is a potent coagonist at NMDA receptors in some mammalian brain areas and possibly involved in the pathogenesis of several psychiatric and neurological disorders, such as schizophrenia (Labrie et al. 2008;Nunes et al. 2012;Balu and Coyle 2015;Ozeki et al. 2016), bipolar disorder (Yamada et al. 2004;Young and Barrett 2015), depression (Hashimoto et al. 2015(Hashimoto et al. , 2016Deutschenbaur et al. 2016), Alzheimer's disease (Paula-Lima et al. 2013;Madeira et al. 2015), and addiction (D'Ascenzo et al. 2014;Seif et al. 2015;Liu et al. 2016). GLY has high affinity for extrasynaptic NMDA receptors, while d-serine has high affinity for synaptic NMDA receptors (Vizi et al. 2013).
There is a paucity of studies on body fluid levels of d-serine in ASD. In 2006, Shinohe et al. (2006) showed that d-serine and l-serine levels in serum were no different between adult patients with ASD and HCs. Comparing with HCs, Tirouvanziam et al. (2011) showed that combined serine levels in plasma were decreased in ASD. In studies on urine samples, Kałużna-Czaplińska et al. (2014) reported that l-serine levels were decreased in ASD, and Noto et al. (2014) reported that l-serine levels were increased, while Evans et al. (2008) and Ming et al. (2012) showed that the combined serine levels were decreased. In the studies in which combined serine levels were reported, the d-serine and l-serine were not measured separately.

Other amino acids
The metabolism of homocysteine is associated closely with folic acid and Vitamin B12 (Ghanizadeh et al. 2012;Desai et al. 2016). Desai et al. (2016) showed that a lack of folic acid may be involved in the pathogenesis of ASD. Bala et al. (2016) reported low plasma levels of Vitamin B12 in ASD compared to values in HCs. James et al. (2004), and Bala et al. (2016) reported that the concentration of homocysteine in plasma with ASD patients is low. However, other studies showed that homocysteine levels were increased in ASD patients compared with HCs: Tu et al. (2012) reported levels in plasma, Ali et al. (2011) and Paşca et al. (2006) reported levels in serum, and Noto et al. (2014) and Puig-Alcaraz et al. (2015) reported levels in urine samples, and all were reported to be increased. Puig-Alcaraz et al. (2015) found that increased urinary levels of homocysteine correlated directly with the severity of deficit in communication skills in ASD.
Arginine is an essential precursor for the synthesis of proteins and nitric oxide, and it can spare GLN, detoxify ammonia, and increase brain blood flow (Good 2011a). Compared with HCs, arginine levels in plasma of ASD patients have been reported to be increased (Kuwabara et al. 2013) or no different (Tirouvanziam et al. 2011). Table 1 shows the reported levels of neuroactive amino acids in patients with ASD in comparison with HCs and includes some other amino acids not mentioned previously in this review (leucine, lysine, citrulline, alanine, valine, isoleucine, threonine, proline, methionine, aspartate, asparagine, phenylalanine, tyrosine, and histidine). Leucine, isoleucine, and valine are all termed branched-chain amino acids (BCAAs) and share a transport system with large, neutral amino acids (LNAAs), such as tryptophan, tyrosine, and phenylalanine which are the precursors of the neurotransmitter amines 5-hydroxytryptamine (5-HT, serotonin) and the catecholamines (Fernstrom 2005). Arnold et al. (2003) reported that the level of the essential amino acids valine, leucine, phenylalanine, and lysine in ASD was 58 % compared to HCs. Although there is a paucity of studies on the levels of BCCAs in ASD, most of the studies report a reduction of BCAA levels in autistic subjects (see Table 1 and the references mentioned therein), suggesting that future research in this area is warranted.

Discussion
Overall, the results on amino acid levels in ASD reported in the literature are, with the possible exception of the BCAAs, inconclusive, and contradictory. Table 1 is a summary of reported differences between ASD patients and HCs in the levels of amino acids. BCAAs are essential amino acids that make up about 1/3 of muscle protein, and these deficiencies may affect muscle and connective tissue integrity in ASD subjects (Evans et al. 2008). It has been suggested that BCAA deficiencies may be related to poor nutrition due to unusual food preferences in ASD children (Arnold et al. 2003).
In studies on amino acids levels in ASD subjects reported in the literature, there has been considerable variation in terms of factors such as age, gender, number of subjects, IQ, and psychoactive medication being taken. Future studies could be improved by standardizing these factors and analyzing levels of several amino acids (including d-serine and BCAAs) simultaneously.

Compliance with ethical standards
Not applicable since this a review of the literature.

Conflict of interest
The authors declare that they have no conflict of interest.
Ethical approval This article does not contain any studies with human or animals conducted by the authors.


s2fieldsofstudy: Biology Medicine

text:
Modulation of IL-6 induced RANKL expression in arthritic synovium by a transcription factor SOX5

Receptor activator of nuclear factor κB ligand (RANKL) is critically involved in bone erosion of rheumatoid arthritis (RA). We previously reported association between younger age at onset of RA and a RANKL promoter SNP that conferred an elevated promoter activity via binding to a transcription factor SOX5. Here we study the regulation of SOX5 levels in relation to RANKL expression in RA synovial fibroblasts (SF) and the development of bone erosion in the collagen-induced arthritis (CIA) mouse. Our data indicated SOX5 levels were higher in synovium and synovial fluid from RA compared to osteoarthritis patients. Pro-inflammatory cytokines upregulated SOX5 and RANKL expression in both primary RA SF and the rheumatoid synovial fibroblast cell line, MH7A. Overexpression of SOX5 resulted in significantly increased RANKL levels, while knockdown of SOX5 resulted in diminished IL-6 mediated RANKL upregulation in MH7A cells. Chromatin immunoprecipitation (ChIP) showed approximately 3-fold enrichment of RANKL-specific DNA in anti-SOX5 immunoprecipitate in IL-6 treated MH7A cells as compared to untreated cells. Locally silencing SOX5 gene significantly diminished RANKL positive cells and bone erosion in CIA mice. These findings suggest SOX5 is an important regulator of IL-6-induced RANKL expression in RA SF.

Receptor activator of nuclear factor κ B ligand (RANKL) is critically involved in bone erosion of rheumatoid arthritis (RA) by enhancing osteoclast formation, function and survival 1 . Increased RANKL levels, locally and systemically, promote excessive osteoclast activity and bone resorption in RA 2 . Targeting RANKL by Denosumab, a fully human monoclonal anti-RANKL antibody, could efficiently prevent bone loss in RA 3 , highlighting the pivotal role of RANKL in mediating bone erosion.
RANKL is expressed on stromal/osteoblastic cells, chondrocytes, activated T, B cells, endothelial cells and synovial fibroblasts (SF) 4 . SF is the main source of RANKL in the inflamed joints of RA 4 . Inflammatory cytokines such as IL-6, IL-1β , TNF-α and IL-17, abundant in the synovial fluid and synovium, are potent inducers of RANKL expression in RA 4 . Binding of RANKL to its unique receptor RANK activates a broad range of signaling cascades, including NF-κ B, MAPK and calcium signaling pathways that could mobilize a series of downstream osteoclast-specific transcription factors such as microphthalmia-associated transcription factor (MITF), c-Fos and nuclear factor of activated T cells c1 (NFATc1) to drive osteoclastogenesis and bone erosion 5 .
To date, how the inflammatory cytokines regulate RANKL gene transcription largely remains unknown. Moreover, although blockade RANKL by monoclonal anti-RANKL antibody Denosumab could efficiently prevent bone loss in resorptive diseases such as RA and osteoporosis, but also could cause rare but significant, clinical complications such as osteonecrosis of the jaw and atypical subtrochanteric femoral fractures 6 , which might due to profound inhibition of RANKL and subsequently affecting bone turnover. In this context, an understanding of how RANKL expression is regulated is therefore important to identify novel therapeutic targets for RA or other resorptive diseases.
We previously reported that a RANKL promoter single-nucleotide polymorphism (SNP) rs7984870 confers an elevated promoter activity after stimulation and is associated with younger age at onset of RA 7,8 . In silico analysis indicated the risk allele of this SNP might create a binding site to SOX5 in the RANKL promoter. We subsequently Scientific RepoRts | 6:32001 | DOI: 10.1038/srep32001 confirmed that SOX5 could bind to the promoter of RANKL gene in Jurkat T cell line after stimulation with TNF-α by electrophoretic mobility shift assay (EMSA) 7 , indicating a potential role of SOX5 in regulation inflammatory cytokine mediated RANKL expression.
SOX5 belongs to the SoxD group of Sox family transcription factor and could be expressed in multiple cell linages including the spermatids, neurons, fetal brain, striated muscles, chondrocytes, B cells [9][10][11][12] . SOX5 functions as transcription factors and primarily plays important roles in the regulation of embryonic development, cell fate, and chondrogenesis 13 . Elevated SOX5 transcripts have been related to invasive growth and reduced apoptosis in tumors and have been associated with progression of certain cancers [14][15][16] . Recent studies have suggested the involvement of SOX5 in B-cell development 9 and Th17 differentiation 17 , suggesting a role of SOX5 in immune responses. Here, we present our findings of the function of SOX5 in RA SF in vitro and in the experimental inflammatory arthritic mice in vivo, supporting this transcription factor SOX5 as an important regulator for cytokine-induced RANKL expression and the development of bone erosion in arthritic joints.

Materials and Methods
Patients and samples. Patients 19 . Clinical characteristics of RA and OA patients are listed in Table S1. The use of human materials was approved by the Institutional Review Board of the First Affiliated Hospital of Nanjing Medical University (Permit Number: Jsph-EC201303114), and written informed consent was obtained from all individuals before the operative procedure. The animal experiments were conducted with the approval of Institutional Animal Care and Use Committee of Nanjing Medical University (Permit Number: IACUC-2014060206). All experiments were carried out in accordance with the approved guidelines.
Immunohistochemistry and Immunofluorescence microscopy. Synovium from patients or mice was snap-frozen and embedded in paraffin for immunohistochemstry or immunofluorescence staining as we previously reported 23 . Rabbit anti-SOX5 (Santa Cruz, sc-20091), mouse anti-RANKL (Abcam, ab22113) and rabbit anti-TRAP (Abcam, ab96372) were used as the primary antibodies for immunohistochemistry. For double-immunofluorescence labeling of human joint tissues, the following primary antibodies were used: rabbit anti-SOX5, mouse anti-TRAP, mouse anti-FGF2 (Abcam, ab8880), mouse anti-CD68 (Abcam, ab125212) and mouse anti-RANKL. Using fluorescence microscopy, cell types were distinguished according to their characteristic morphology and confirmed by immunoreactivity with anti-FGF2 (FLS), anti-CD68 (macrophages) and anti-TRAP (osteoclast) antibodies.
Confocal Microscopy. MH7A were treated with or without IL-6 (100 ng/ml) for the indicated time interval and fixed with 100% methanol and then washed with PBS and incubated with the rabbit polyclonal SOX5 antibody and/or the mouse monoclonal RANKL antibody. Cells were washed with PBS and incubated with rabbit anti-mouse IgG-Rhodamine conjugated or goat anti-rabbit IgG-FITC conjugated. Confocal microscopy was performed on an LSM-510 laser scanning microscope (Olympus, Center Valley, PA). Final images were processed using Adobe Photoshop software.

Construction of shSOX5, lentivirus-shSOX5 and recombinant Adenovirus SOX5 expression vectors.
For construction of shSOX5, two different siRNA sequences that silenced SOX5 effectively were used in this study (Table S2). The complementary DNA oligonucleotides were subcloned into the hairpin siRNA expression vector pRNAT-U6.1/Neo (Invitrogen). For local silencing SOX5 in CIA mice joint, the recombinant lentiviral vector hU6-MCS-CMV-eGFP was used to construct lentivirus-SOX5 shRNA as previously described 24 . For construction of SOX5 overexpression vector, the full-length of SOX5 cDNA was generated via standard PCR and inserted into the pAd-CMV-MC5 as described 24 . All the cloned sequences were confirmed by DNA sequencing.
Overexpression and knockdown experiments. For silencing SOX5, 1 μ g/ml of shSOX5 was transfected into MH7A for 24 h using X-treme GENE HP DNA Transfection Reagent (R&D, 6365787001) and then treated with IL-6 for 24 h. Non-targeting shRNAs served as negative controls (described as mock in the current study). For SOX5 overexpression experiments, MH7A was transduced with 10 7 pfu/ml of Ad-SOX5 for 96 h. Control transfectants were obtained by transduction with Ad-EGFP.
Chromatin immunoprecipitation (ChIP). ChIP assays were performed essentially as described 25 . In brief, MH7A were treated with or without IL-6 (100 ng/ml) for 24h and cross-linked with 1% formaldehyde for 10 minutes and then lyzed inlysis buffer containing with protease inhibitor (Millipore). DNA was fragmented into ~200bp pieces using a Branson 250 sonicator. Each ChIP sample containing 100 μ g nucleoprotein was used for the immunoprecipitation reaction with anti-SOX5 and nonspecific IgG, and 10% of the pre-cleared chromatin was set aside as input control. Purified DNAs were subjected to quantitative PCR (qPCR) using the primer sets listed in Supplementary Table S3. Fold enrichment of the targeted genomic sequences were calculated over IgG.

Real-Time PCR and Western Blotting. Gene expression was quantified by SYBR Green real-time PCR
using an ABI Prism 7900 Sequence Detection System. The sequences of the primers are listed in Table S3. Relative expression was calculated with normalization to β -actin values by using the 2−Δ Δ ct method. The following antibodies were used for Western blotting: rabbit polyclonal anti-SOX5 antibody (Millpore, ABN513); goat anti-rabbit RANKL antibody (Abcam); rabbit polyclonal anti-β -Actin antibody (Cell Signaling Technology, 8H10D10).
Induction of collagen-induced arthritis in DBA/1J mice and histological assessment. The induction of CIA was described previously 26 . Briefly, male 6 wk-old DBA/1J (purchased from purchased from the Shanghai Laboratory Animal Center, Chinese Academy of Science) mice were injected intradermally at the base of the tail with 200 μ g of bovine CII (Chondrex, 20021) emulsified with complete Freund adjuvant, and their arthritis manifestations were graded and scored as described 26 . For standard histologic assessment, hind paws were fixed overnight in 4% paraformaldehyde and decalcified using EDTA. The material was then embedded in paraffin, cut and 1-2 μ m sections were stained with H&E. The histological arthritis score was determined in a blinded fashion for changes in synovial lining, cellular infiltrate, cartilage damage, and pannus, as previously described 27 . ELISA. Concentrations of SOX5 levels in synovial fluids of RA and OA patients were quantified using a commercial ELISA kit (Huamei, Wuhan, China, 20110647) according to the manufacturer's recommendations. Briefly, synovial fluids samples (1: 5 dilution) and standards were added to the 96-well plates. After incubation for 2 hours and washing 5 times, mouse SOX5 conjugate was added, followed by incubation with TMB substrate solution and stop solution. The intensity of the color reaction was measured by a microplate reader at a wavelength of 450 nm. Concentrations of SOX5 were determined by a standard curve according to the manufacturer's instructions.

Micro-computed tomography (CT) analyses. Computed tomographic images of the knee joints and
paws of the mice in all three groups (n = 5) were acquired on day 42, using a micro-CT Scan SkyScan1176S scanner at a resolution of 9 μ m. Image acquisition required 6 min using the following parameters: 50 kV, 500 μ A, 0.5 mm aluminium filter, 180° scan, rotation step 0.7° and frame averaging of 1. For verification of bone destruction, 3-dimensional models of the knee joints and paws were reconstructed using SkyScan CT Analyzer version 1.8.

Osteoclast differentiation and TRAP staining.
To assess the effect of SOX5 on osteoclastogenesis, RAW264.7 cells were plated at a density of 2 × 10 4 cells/well in a 12-well culture plate, and transfect with mock or Lv-siSox5 for 48 h and then cultured with or without 100 ng/ml RANKL plus 20 ng/ml M-M-CSF; After 3-5 d, cells were stained for TRAP using an Acid Phosphatase, Leukocyte (TRAP) Kit (Sigma) according to the manufacturer's instructions. TRAP-positive multinucleated cells with three or more nuclei were counted as osteoclasts under a light microscope.
Statistical analysis. Data were presented as mean and SD. Data were firstly tested for conformation to a normal distribution using the Shapiro-Wilk test and then were analyzed by Student's t test (pair-wise comparisons) or analysis of variance (ANOVA), as appropriate. P values less than 0.05 were considered significant.

Results
Increased expression levels of SOX5 in RA synovium. We previously reported that SOX5 could bind to the promoter of RANKL gene in Jurkat T-cells after stimulation 7 . To explore possible functions of SOX5 in RA pathogenesis, we firstly compared the levels of SOX5 expression between RA and osteoarthritis (OA) patients. Real-time PCR revealed higher expression levels of SOX5 relative to β -actin in synovium of RA patients (n = 20) than those in OA counterparts (n = 20) (Fig. 1A, p = 0.001). We further examined protein levels of SOX5 in synovial fluids from RA (n = 30) and OA patients (n = 27). Similar to the expression in synovium tissue, SOX5 was increased approximately 2-fold in RA synovial fluids (Fig. 1B, p = 0.001) compared with those in OA patients. Immunohistochemical staining indicated SOX5 positive cells were more abundant in RA synovium samples than those of OA samples (n = 5 each) (Fig. 1C,F).
We extended these results using the CIA DBA/1J mice. Consistent with the expression in human RA synovium, real-time PCR (Fig. 1D) and immunohistochemical staining (Fig. 1E,F) revealed higher expression of Sox5 in inflamed synovium from CIA mice as compared with normal control mice (NC).

Pro-inflammatory cytokines induce SOX5 and RANKL expression in RASF. Given that SOX5
was up-regulated both in lining cells and sublining cells of synovium in RA patients (Fig. 1C), which primarily consists of SF and macrophages, we determined which cell type expresses SOX5 in inflamed RA synovium. Double-immunofluorescence studies were used to investigate co-localization of SOX5 and markers for macrophages (CD68), SF (FGF) and osteoclasts (TRAP). More SOX5-positive cells could be detected in SF (Fig. 2A, middle and Fig. 2B) than in macrophages ( Fig. 2A, upper and Fig. 2B), whereas co-localization between TRAP positive cells and SOX5 was only occasionally observed in RA synovium ( Fig. 2A, lower and Fig. 2B). These data indicate that SOX5 is mainly expressed in SF of RA synovium.
Scientific RepoRts | 6:32001 | DOI: 10.1038/srep32001 Next, we investigated the mechanisms that could induce high expression of SOX5 in RA SF. Human rheumatoid fibroblast-like synoviocyte MH7A cell line were treated with inflammatory cytokines IL-1β , TNF-α , IL-6 or IL-17 for 0-36 h. Real-time PCR indicated that IL-1β , TNF-α and IL-6 could induce up-regulation of SOX5 in MH7A cell line at 24-36h (Fig. 2C, left). As expected, the expression levels of RANKL were concomitantly increased in MH7A (Fig. 2C, right) in response to the pro-inflammatory cytokines. Among these cytokines, IL-6 showed the robust effect on inducing SOX5 and RANKL expression at 24h, whereas IL-17 failed to drive SOX5 expression (data not shown). The similar results were confirmed at primary RA SF upon these inflammatory cytokines stimulation for 24 h (Fig. 2D, left for SOX5 and right for RANKL expression. Consistent with mRNA expression, western blot analysis showed that the protein levels of SOX5 and RANKL were concomitantly increased in MH7A after stimulation (Fig. 2E). These findings suggest that inflammatory cytokines upregulate SOX5 in SF, which is accompanied by increased RANKL levels.
Co-localization of SOX5 and RANKL in RA SF. We further examined SOX5 and RANKL expression in synovial tissue from RA patients by immunofluorescence staining. We found that SOX5 co-localized with RANKL in the inflamed RA synovium (Fig. 3A). We further assessed the effect of pro-inflammatory SOX5 and RANKL expression in RA SF by immunofluorescence staining. Our results (Fig. 2C,D) indicated that IL-6 was the strongest inducer of SOX5 and RANKL expression in SF at 24 h, and then we confirmed this result at 48-72 h (data not show). So, IL-6 was used to stimulate MH7A cells in the next experiment. Although did not reach a statistical differences, we found an increased tendency of co-localization of SOX5 and RANKL on MH7A cell surface at 0 to 60 min after IL-6 stimulation by confocal microscopy (Fig. 3B,C), implying IL-6 could prompt co-localization of SOX5 and RANKL in RA SF.

SOX5 knockdown by shRNA reduce IL-6 -mediated RANKL expression in RA SF.
We assessed the effect of SOX5 knockdown on IL-6 induced RANKL mRNA expression in RA SF using shRNA targeting SOX5. MH7A was transfected with SOX5-shRNA to silence SOX5 expression. As shown in Fig. 4A, SOX5-shRNA transfection resulted in an approximately 51% decrease in SOX5 expression by MH7A cells, compared with mock transfected group. Consistent with Fig. 2D, RANKL expression was markedly increased in MH7A upon IL-6 stimulation. However, IL-6-induced RANKL expression was significantly downregulated in SOX5-shRNA treated MH7A in comparison with mock-transfected controls (Fig. 4B, p < 0.05), suggesting that SOX5 is required for IL-6-induced RANKL expression in RA SF. Overexpression of SOX5 regulates expression of RANKL in RA SF. We asked whether SOX5 could directly regulate RANKL expression by overexpressing SOX5 in MH7A. As shown in Fig. 4C,D, transfection with adenovirus vectors expressing SOX5 (Ad-SOX5) resulted in overexpression of SOX5 in MH7A cells with concomitantly increased RANKL levels (Fig. 4D, p < 0.001), comparing to those transfected with EGFP-expression vector controls. Osteoprotegerin (OPG) is a decoy receptor for RANKL. The levels of OPG was also determined in SOX5 overexpressed and silenced MH7A cells. However, transfection with Ad-SOX5 or SOX5-shRNA did not affect OPG expression in MH7A ( Figure S1). Taken together, these findings strongly suggest that SOX5 plays an important role in the regulation of RANKL expression.

IL-6 facilitates the binding of SOX5 to RANKL promoter in SF.
We have previously shown that SOX5 could bind to genomic sequences within the RANKL promoter in Jurkat cells using EMSA assay; moreover, luciferase study indicated RANKL promoter activity could be markedly decreased after mutation of SOX5 binding site (−1857) in RANKL promoter, implying SOX5 might function as an activator of the human RANKL promoter 7 . To further elucidate interaction between the transcriptional factor SOX5 and RANKL promoter, a ChIP assay was performed. A markedly higher amount of chromatin containing RANKL promoter region was immunoprecipitated by anti-SOX5 antibody compared with control IgG in RA SF (Fig. 4E). We then addressed whether IL-6 stimulation could promote the binding of SOX5 with RANKL in SF. We analyzed SOX5 immunoprecipitates by qPCR for the enrichment of RANKL-specific DNA in IL-6 treated MH7A cells. As shown in Fig. 4F, MH7A treated with IL-6 induced approximately 3-fold enrichment of RANKL-specific DNA in anti-SOX5 immunoprecipitate as compared to untreated cells. These results indicate that IL-6 could facilitate the binding of SOX5 to RANKL promoter in SF.

Intra-articular administration of Lentivirus expressing shRNA for Sox5 ameliorates bone erosion in CIA mice in part by suppression Rankl expression.
We demonstrated the presence of increased SOX5 in RA synovium and that SOX5 is critical for IL-6-induced RANKL expression in vitro. Next, we investigated the possible in vivo functions of Sox5 in the CIA model. We locally knocked down Sox5 expression in CIA mice by injecting lentiviral vectors expressing siRNA (Lv-siSox5) intra-articularly into the hind ankle at day 1 of the second immunization. Administration of Lv-siSox5 into joints of CIA mice effectively decreased Sox5 mRNA in the joint tissue as compared with mock siRNA injection (Fig. 5A, p = 0.03). Consistent with mRNA expression, Sox5 protein level was significantly decreased in the joint tissue from Lv-siSox5-treated CIA mice (Fig. 5B) in comparison with PBS treated or mock siRNA transfection. CIA incidence was decreased by almost 50% after Lv-siSox5 treatment ( Figure S2A); accordingly, the arthritis score ( Figure S2B) and articular swelling ( Figure S2C) were significantly decreased. Consistent with the inhibitory effects of RANKL expression in vitro, local silencing of Sox5 markedly decreased Rankl mRNA expression (Fig. 5C, p = 0.04) in the arthritic joint from CIA mice. Immunohistochemstry staining indicated that RANKL protein expression were diminished in synovium samples from Lv-siSox5-treated CIA mice (Fig. 5D,E) compared with mock-treated mice. Histological analysis (Fig. 5F) indicated that local silencing of Sox5 in joint tissues significantly inhibited synovitis, synovial hyperplasia and bone erosion in CIA mice.
Micro-CT confirmed a significant decrease in bone loss in the periarticular bone of paws and ankles from Lv-siSox5 treated CIA mice (Fig. 6A,B). We quantified the bone microarchitecture values of bone volume, trabecular thickness, trabeculae number and bone mineral density. Levels of bone volume, trabeculae number and bone mineral density was statistically significant decreased in Mcok treated CIA mice. However, bone volume, and trabeculae number and bone mineral density was increased in Lv-siSox5 treated CIA mice as compared with Mock treated mice (Fig. 6C).
Osteoclast is the unique cell that responsible for bone erosion in RA. We addressed whether SOX5 can directly stimulate osteoclast formation independent of RANKL. Osteoclast precursor cell line RAW264.7 was transfected with Lv-siSox5 for 48h and then were treated with 150 ng/ml RANKL and 30 ng/ml M-CSF for 3-5 d to induce osteoclast differentiation. Silencing Sox5 did not affect the formation of TRAP-positive cells in osteoclast precursor cell treated with RANKL. However, the multinucleated TRAP staining positive osteoclast-like cells were not found in Lv-siSox5 transfected RAW264.7 in the absence of RANKL, implying that SOX5 mediated bone erosion is depended of RANKL (Fig. 6D). Collectively, these results indicate that local suppression Sox5 expression ameliorates arthritis and bone loss in CIA mice likely by inhibiting Rankl expression.

Discussion
It is well established that the bone damage in RA is caused by inflammation-induced abnormal expression of RANKL to drive osteoclastogenesis 2 . The downstream signals required for RANKL activation and osteoclast differentiation have been extensively studied 28 . However, there is limited understanding of how the pro-inflammatory cytokines regulate transcription of the human RANKL gene 28 . Our current findings identify SOX5 as an important regulator of proinflammatory cytokine-induced expression of RANKL in RA. To our knowledge, this is the first study providing evidence for high expression of SOX5 in synovium samples of RA patients and CIA model. Our data demonstrate that knockdown of SOX5 abrogated the stimulatory effects of IL-6 on RANKL expression in SF in vitro. Local Sox5 gene silencing significantly ameliorated clinical symptoms and bone erosion in CIA mice, which is likely mediated by suppressing Rankl signaling. Collectively, these results provide novel insights into the regulation of RANKL expression in inflamed synoviocytes of RA patients by SOX5.
SOX proteins predominantly function as transcriptional activators by differential binding to particular flanking sequences of target genes or by their interactions with other transcription factors 28 . To date, little is known about SOX5 in RA. Our previous EMSA experiment showed that SOX5 could bind to RANKL promoter in Jurkat cells after stimulation with TNFα 7 , providing direct evidence of an interaction between SOX5 and RANKL. In current study, we validated that SOX5 was able to directly bind to the promoter of RANKL by ChIP assay. We determined the direct effect of SOX5 on RANKL transcript in RA SF. We found in vitro knockdown the SOX5 significantly decreased IL-6-induced RANKL expression in SF, implying that SOX5 is required for pro-inflammatory induced RANKL expression.
Importantly, our data indicated that SOX5 only showed weak binding activity with RANKL in unstimulated SF; however, this binding activity was markedly enhanced after IL-6 treatment in the RA synovium derived cell line, MH7A in our ChIP assay. This result is reinforced by the concomitantly increased SOX5 and RANKL levels in primary RA SF and MH7A cell line after stimulation with IL-6. Taken together, it is possible that IL-6 promote SOX5 expression and thereby enhance the binding of SOX5 to RANKL, and the increase in SOX5 binding to the RANKL promoter at least in part account for the much larger increase in RANKL expression in RA SF.
IL-6 plays a key role in driving osteoclastogenesis and bone resorption in RA. Previous study indicate IL-6 does not induce osteoclast formation per se, but could mediated it by a direct effects in which IL-6 engages IL-6R directly on osteoclast precursors or by an indirect effect on prompting RANKL expression in mesenchymal cells 29 . Our data shed a new light on that IL-6 mediated regulation of inflammatory bone erosion in RA by an indirect effect on prompting transcriptional factor SOX5 binding to RANKL.
These results are further supported by our in vivo findings that local Sox5 gene silencing results in markedly bone erosion in CIA mice, which could be attributed in part to the concomitant Rankl down-regulation. RANKL plays a crucial role in osteoclast formation, function and survival. Our data indicate blocking endogenous Sox5 did not affect osteoclast formation in the presence with RANKL; however, Lv-siSox5 transfection is incapable of driving osteoclastogenesis in osteoclast precursor cell in the absence of RANKL, highlighting that SOX5 mediated bone erosion is depended of RANKL.
To date, transcriptional regulatory regions of mouse Rankl has been well studied, but the regulatory motifs in human RANKL remains largely unclear 30,31 . Our previous data showed that SOX5 could bind to the −1857 site of the RANKL promoter 7 . However, whether one or multiple SOX5 binding sites could regulate RANKL expression is beyond the scope of the current study. The transcriptional regulatory mechanism of SOX5 on RANKL should be further clarified in the future. Our data indicated that CIA incidence and arthritis score was decreased after Lv-siSox5 treatment. Although our data provide solid evidence that SOX5 is critically involved in RA bone erosion by modulating the RANKL pathway, we can't exclude a potential anti-inflammatory role of SOX5 contribute to improved bone erosion in CIA in current study. Further study also need to clarify the effect of SOX5 on inflammation process in CIA mice.
In summary, our findings establish a key role for SOX5 in inflammation-mediated RANKL expression in arthritic joint. Given that extensive inhibit of RANKL expression could affect bone turnover, our study positions selective blockage of SOX5 as a potential novel therapeutic target for RA and possibly other bone resorptive diseases, such as osteoporosis.


s2fieldsofstudy: Biology Chemistry

text:
miR-194 inhibits the proliferation, invasion, migration, and enhances the chemosensitivity of non-small cell lung cancer cells by targeting forkhead box A1 protein

Recent studies have implied that miRNAs may play a crucial role in tumor progression and may be involved in the modulation of some drug resistance in cancer cells. Earlier studies have demonstrated that miR-194 was involved in tumor metastasis and drug resistance in non-small cell lung cancer (NSCLC), whereas their expression and roles on NSCLC still need further elucidation. In the current study, we found that miR-194 is decreased in NSCLC samples compared with adjacent non-cancerous lung samples, and low expression of miR-194 predicts poor patient survival. Both in vitro and in vivo experiments showed that ectopic stable expression miR-194 suppressed proliferation, migration, invasion and metastasis and induced apoptosis in NSCLC cells and that this suppression could be reversed by reintroducing forkhead box A1 (FOXA1), a functional target of miR-194. In addition, miR-194 was downregulated in in cisplatin-resisted human NSCLC cell line-A549/DDP and overexpression of miR-194 increases cisplatin sensitivity. These findings suggested that miR-194 inhibits proliferation and metastasis and reverses cisplatin-resistance of NSCLC cells and may be useful as a new potential therapeutic target for NSCLC.

INTRODUCTION
Non-small cell lung cancer (NSCLC) is one of the most common cancers and one of the leading causes of cancer related mortality across the world [1]. Most common pulmonary malignancy are NSCLC, it accounts for 85% of cases of pulmonary malignancies [2]. The most effective treatment for NSCLC is surgical resection, but most of patients diagnosed with NSCLC have reached an advanced pathological stage, at which point the patients miss the best time for surgical resection. Despite significant advances in surgery, chemotherapy and radiotherapy, the 5-year survival rate for lung cancer was only 15%. Therefore, new treatment strategies to identify new molecular targets for prevention and treatment of NSCLC are urgently needed.
MicroRNAs (miRNAs) are a class of single-stranded noncoding RNA molecules of 18-24 nucleotides in length.
They play a key role in many biological processes through negative regulation of the expression of target genes posttranscriptionally by sequence-specific binding to the 3′ untranslated regions (UTRs) of specific mRNA targets [3].
MiR-194 is a vertebrate specific microRNA with a known role in mitochondrial energy production [4], inhibition of inflammation [5], chondrogenesis [6] and neuronal differentiation [7]. It has also been implicated in a number of malignancies, including oral squamous cell carcinoma [8], breast cancer [9,10] and so on. Another study showed that miR-194, via its actions on the protein FOXM1, inhibits gastric carcinoma cell migration, invasion, and epithelial to mesenchymal transition [11]. In oral squamous cell carcinoma, miR-194 appears to medicate PI3K-Akt-FOXO3a signaling pathway via its negative regulation of AGK [8]. A recent study reported that miR-194 was decreased and associated with tumor size and tumor differentiation in colorectal cancer, overexpression of miR-194 suppresses tumor growth by regulating the MAP4K4/c-Jun/MDM2 signaling pathway. MiR-194, commonly repressed in colorectal cancer, suppresses tumor growth by regulating the MAP4K4/c-Jun/MDM2 signaling pathway. Moreover, miR-194 was found to reduce in primary tumor samples with metastasis and enforcing miR-194 suppressed motility and invasiveness of lung cancer cells in vitro and in vivo through downregulation of two key functional factors, BMP1 and p27 kip1 . miR-194 suppresses metastasis of non-small cell lung cancer through regulating expression of BMP1 and p27 kip1 . However, the roles of miR-194 in NSCLC growth and metastasis and the molecular mechanism remain to be investigated.
FOXA1 is a member of the human Forkhead-box family. These genes have been implicated in congenital disorders, diabetes, and carcinogenesis [12]. In squamous cell carcinoma of the lung, FOXA1 expression has been shown to be associated with distant metastases and poorer overall survival [13]. It has also been shown to promote epithelial to mesenchymal transition (EMT) in NSCLC, perhaps explaining its association with the propensity to metastasize in NSCLC cells [14]. More recently, FOXA1 was found to upregulate and high FOXA1 expression have lower rates of progression free survival in Urothelial carcinoma of the bladder. MicroRNA-99a and 100 mediated upregulation of FOXA1 in bladder cancer.
In this study, we first determined the miR-194 expression in NSCLC tissues and their corresponding adjacent normal tissues, then investigated the functional role of miR-194 in tumourigenesis, metastasis, and apoptosis induction in NSCLC cells. We also provide experimental evidence that miR-194 regulated cellular function via directly interacting with the FOXA1 mRNA at the 3′-UTR. In all, our data supports the notion that miR-194 acts as a tumor suppressor and might be a novel potential therapeutic target for NSCLC.

miR-194 was significantly downregulated and correlated with poor prognosis
Expression levels of miR-194 were determined in 64 pairs of NSCLC tissue and paired adjacent non-tumor tissue. Expression of miR-194 in NSCLC tumor tissue was significantly lower than in the paired non-tumor tissue (p < 0.01) ( Figure 1A). Expression of miR-194 was also examined in NSCLC tissues of varying stage. In higher stage lesions (stage III-IV), miR-194 expression was significantly lower than in lower stage lesions (stage I-II) (p = 0.0004) ( Figure 1B). Furthermore, we investigated the potential associations between miR-194 expression and patients' clinicopathological variables. Clinicopathological variables of NSCLC patients were shown in Table 1. Interestingly, low miR-194 expression was significantly correlated with Lymph node metastasis and TNM stage (P < 0.05). Overall survival was examined in patients with NSCLC's expressing varying amounts of miR-194. 29 patients had tumors that expressed high levels of miR-194, while 35 patients had tumors that expressed low levels of miR-194. Patients with tumors that expressed high levels of miR-194 had significantly longer overall survival than patients who had tumors that expressed low levels of miR-194 (p = 0.0002) ( Figure 1C). Finally, expression levels of miR-194 were determined in six NSCLC cell lines, with the benign human bronchial epithelial cell line (16HBE) serving as a control. Expression levels of miR-194 were significantly less in all of the NSCLC cell lines compared to the control (p < 0.01) ( Figure 1D), especially, NCI-H1299 and A549 cells showed lowest miR-194 levels. Overall, these results indicated that not only does decreased expression of miR-194 distinguish benign tissue from malignant NSCLC but also that the magnitude of the decrease in expression in tumor tissue can characterize the aggressiveness of the tumor.

miR-194 inhibits NSCLC cell proliferation both in vitro and in vivo
With the knowledge that lower level of miR-194 is associated with more aggressive NSCLC behavior, we then attempted to determine what factors contribute to the increase in aggressiveness of these tumors. We therefore examined effect of miR-194 on cell proliferation. Transfection of the H1299 and A549 NSCLC cell lines with either a miR-194 vector or an empty control vector was performed. Increased expression of miR-194 in the cells transfected with the miR-194 vector was confirmed by qRT-PCR in both cell lines (p < 0.01) (Figure 2A). The MTT assay was then performed on cells from both lines to assess cell viability. Both H1299 and A549 cells that were transfected with the miR-194 vector demonstrated significantly reduced cell viability compared to H1299 and A549 cells transfected with the control vector respectively after three days (p < 0.01) ( Figure 2B). The colony formation assay was also performed to assess proliferative capacity. Again, both H1299 and A549 cells that were transfected with the miR-194 vector exhibited significantly lower rates of colony formation than H1299 and A549 cells transfected with the control vector respectively (p < 0.01). Representative micrographs of this assay are also provided ( Figure 2C). Finally, in vivo analysis of tumor growth was assessed by measuring both tumor volume and weight. A549 cells that were transfected with the miR-194 vector and implanted into nude mice grew into both significantly smaller (p < 0.01) ( Figure 2E) and lighter (p < 0.01) ( Figure 2F) tumors than A549 cells that were transfected with the empty control vector and implanted into nude mice. Gross specimens dissected from the mice pictorially demonstrate this difference in volume ( Figure 2D). Overall, these results suggested that miR-194 has an inhibitory effect on tumor aggressiveness by reducing cell proliferation both in vitro and in vivo.

miR-194 arrests the cell cycle and induces apoptosis in NSCLC cells
The reduction in cell proliferation seen in NSCLC cells treated with miR-194 was then further examined by looking at its effect on apoptosis and the cell cycle. The FITC and PI apoptosis assays were used to determine rates of apoptosis in the various cells. Both H1299 and A549 cells that were transfected with the miR-194 vector exhibited significantly increased rates of apoptosis compared to H1299 and A549 cells transfected with the control vector respectively (p < 0.01) ( Figure 3A). A cell cycle assay was subsequently performed to determine the percent of cells in each phase of the cell cycle. A significantly lower percent of both H1299 and A549 cells transfected with the miR-194 vector were in S phase than H1299 and A549 cells transfected with the control vector respectively (p < 0.01) ( Figure 3B). Conversely, a significantly higher percent of both H1299 and A549 cells transfected with the miR-194 vector were in G2 phase than H1299 and A549 cells transfected with the control vector respectively (p < 0.01) ( Figure 3B). These findings suggested that miR-194 reduces cellular proliferation in NSCLC tissue both by increasing apoptosis as well as by inhibiting cellular mitosis.
Finally, expression levels of specific proteins known to be involved in the regulation of apoptosis were determined by Western blot. Both H1299 and A549 cells that were transfected with the miR-194 vector expressed greater levels of Bax and lower levels of BCL-2 and CyclinD1 than H1299 and A549 cells transfected with the control vector respectively ( Figure 3C). As Bax is a protein necessary for the initiation of apoptosis, its increased expression in cells treated with the miR-194 vector confirms the association between miR-194 expression and apoptosis. Conversely, as BCL-2 inhibits apoptosis and Cyclin D1 promotes progression through the cell cycle, their reduced expression in cells treated with miR-194 vector also confirm the association between miR-194 expression and apoptosis.

miR-194 inhibits NSCLC migration and invasion both in vitro and in vivo
Cell proliferation is an important measure of tumor aggressiveness, though it is an incomplete measure. A tumor's ability to metastasize is a vital component of its aggressiveness and is directly related to its migratory and invasive capacity. Therefore, migration and invasion assays were performed. The numbers of invasive and migratory cells per high power field were counted. Both H1299 (p < 0.01) ( Figure 4A) and A549 (p < 0.01) ( Figure 4B) cells that were transfected with the miR-194 vector exhibited significantly reduced rates of migratory and invasive capacity compared to H1299 and A549 cells transfected with the control vector respectively.
Given that NSCLC cells treated with the miR-194 vector exhibited reduced migratory and invasive capacity in vitro, we therefore examined the metastatic capacity of these cells in vivo. Thin histologic sections of the lungs were obtained from mice that had been implanted with tumor cells. The number of metastatic nodules were counted. Mice that had been implanted with A549 cells  treated with the miR-194 vector had a significantly lower number of pulmonary metastases than mice that had been implanted with A549 cells treated with the control vector (p < 0.01). Representative micrograph sections from the dissected lungs are shown ( Figure 4C).

FOXA1 is a direct target of miR-194
Because miR-194 expression is associated with both decreased tumor growth and metastatic capacity, a search for a potential binding protein was performed. FOXA1 is one such protein, TargetScan demonstrated a putative binding site to miR-194 in the 3' UTR region of FOXA1 (FOXA1-WT) ( Figure 5A). A mutated FOXA1 construct (FOXA1-WT) was also created, with mutations at the binding site ( Figure 5A). A luciferase assay was subsequently performed on H1299 cells transfected with either the miR-194 vector or the control vector and subsequently treated with either the FOXA1-WT protein or the FOXA1-MUT protein. Cells co-transfected with the miR-194 vector and the FOXA1-WT protein had significantly lower levels of luciferase activity than cells co-transfected with the control vector and the FOXA1-WT protein (p < 0.01) ( Figure 5B). Conversely, cells co-transfected with the miR-194 vector and the FOXA1-MUT protein had similar levels of luciferase activity as cells co-transfected with the control vector and the FOXA1-MUT protein ( Figure 5B).
Quantitative real-time PCR and Western blot were then used to determine expression levels of native FOXA1 in cells transfected with the miR-194 vector. Both H1299 (p < 0.01) and A549 (p < 0.01) cells that were transfected with the miR-194 vector had significantly lower levels of FOXA1 mRNA than H1299 and A549 cells transfected with the control vector respectively ( Figure 5C). Subsequently, cells transfected with the miR-194 vector also had reduced expression of FOXA1 ( Figure 5D). FOXA1 mRNA levels were also measured in non-tumor tissue. NSCLC tissue had significantly higher levels of FOXA1 mRNA than paired non-tumor tissue (p < 0.01) ( Figure 5E). Further, Pearson correlation analysis demonstrated an inverse relationship between FOXA1 mRNA levels and miR-194 levels (p < 0.001) ( Figure 5F). Overall, the above findings suggest that binding of miR-194 to FOXA1 results in the degradation of the FOXA1 protein. When miR-194 levels are low, as in NSCLC tissue, FOXA1 levels remain elevated.

Expression of FOXA1 could partially restore the pro-apoptotic and anti-invasion function of miR-194
Having confirmed that miR-194 acts to reduce FOXA1 levels in NSCLC cells, we then wished to determine whether this action of miR-194 is responsible for its tumorigenic effects. We therefore reintroduced FOXA1 expression in NSCLC cells that overexpressed miR-194 and determined its effect on apoptosis and metastatic capacity. Overexpression of FOXA1 significantly reduced the rates of apoptosis in NSCLC cells. Both H1299-miR-194 (p < 0.01) and A549-miR-194 (p < 0.01) cells that were induced to overexpress FOXA1 demonstrated reduced rates of apoptosis compared to the control ( Figure 6A). Overexpression of FOXA1 also restored both the migratory and invasive capacities of NSCLC cells transfected with miR-194. Both H1299-miR-194 (p < 0.01) and A549-miR-194 (p < 0.01) cells that were induced to overexpress FOXA1 exhibited significantly increased capacities to both migrate and invade compared to the control ( Figure 6B). Finally, western blot was used to confirm anti-apoptotic action of FOXA1. Both H1299-miR-194 and A549-miR-194 cells overexpressing FOXA1 expressed greater levels of the anti-apoptotic proteins BCL-2 and Cyclin D1 and recued levels of the proapoptotic protein Bax compared to control ( Figure 6C). These findings suggest that FOXA1 is responsible for the tumorigenic effects in NSCLC cells, and reduction of FOXA1 via miR-194 inhibits those effects.

miR-194 regulates the sensitivity of NSCLC cells to DDP in vitro
Finally, we wished to assess the sensitivity of NSCLC cells to chemotherapy based on their expression level of miR-194. We first examined miR-194 expression in cisplatin-resistant NSCLC cells (A549/DPP). A549/ DPP cells expressed significantly lower levels of miR-194 than A549 cisplatin-sensitive cells (p < 0.01) ( Figure 7A). We then examined miR-194 expression in A549/DPP cells transfected with the miR-194 vector versus the control vector. These cells expressed significantly greater levels of miR-194 than the control (p < 0.01) ( Figure 7B), thus confirming the efficacy of the vector in the cisplatinresistant line. Next, we examined the half maximal inhibitory concentration of DPP for A549/DPP cells transfected with the miR-194 vector. These cells had a significantly lower half maximal inhibitory concentration (IC50) than cells transfected with the control vector (p < 0.01) ( Figure 7C). Lastly, we determined the rate of apoptosis in A549/DPP cells transfected with the miR-194 vector. These cells exhibited a significantly higher rate of apoptosis than the control (p < 0.01) ( Figure 7D). Overall, these findings suggest that increased expression of miR-194 improves the sensitivity of NSCLC cells to the chemotherapeutic agent DPP, whereby cells that express higher levels of miR-194 not only have higher rates of apoptosis but also require a significantly smaller amount of the drug to produce the same effect.
In this study, we have demonstrated a mechanism that miR-194 was decreased in clinical NSCLC tissues compared to adjacent normal lung tissues and that miR-194 played a critical role in NSCLC progression by regulating cell proliferation and invasion via negatively regulate FOXA1. The roles of miR-194 in this study were consistent with other studies, which showed significant growth inhibition in several cancer cell types [34][35][36][37]. In gastric cancer, overexpression of miR-194 was associated with decreased tumor size and less advanced tumor stage, though was not associated with cell proliferation [19]. Yet another study examining miR-194's role in preventing the progression of gastric cancer suggested that it was miR-194's regulation of the protein RBX1 that was responsible for its inhibitory effect. In endometrial cancer, miR-194 was also found to have a protective effect, increasing levels of E-cadherin and reducing levels of vimentin, thus inhibiting cellular invasion via its regulation of the  protein BMI-1 [21]. Patients with endometrial cancers that produced higher levels of miR-194 also had a better prognosis [22]. In renal cell carcinoma (RCC), miR-194 overexpression results in reduced migratory and invasive capacity of RCC cells [23]. In osteosarcoma, overexpression of miR-194 inhibits tumor growth and metastatic potential via the regulation of CDH2 and IGF1R [27]. In colorectal carcinoma, miR-194 inhibits cell viability and invasion via its actions on the AKT2 pathway. It is also expressed in lower amounts with increasing tumor grade [28]. Another study had similar findings regarding the role of miR-194 in colorectal carcinoma, though it suggested that miR-194 regulated the MAP4K4/c-Jun/MDM2 signaling pathway [29]. In my present study, overexpression of miR-194 inhibited cell proliferation, migration, and invasion in vitro and in vivo. These data consisted with previous results that miR-194 inhibits the capacity of NSCLC cells to metastasis [30]. And our data also showed that miR-194 was decreased in NSCLC tissues and associated with poor prognosis.
Cisplatin resistance is still a major obstacle for the clinical therapy of NSCLC with advanced stage. Our data showed that miR-194 was significantly decreased in stage III-IV. It is interesting to investigate whether miR-194 would affect the NSCLC cells sensitive to DDP. As we expected, overexpression of miR-194 are more prone to apoptosis when treated with a DDP and require a significantly reduced half maximal inhibitory concentration of the chemotherapeutic agent. Our findings suggest that miR-194 could play an important role in the development of cisplatin resistance in NSCLC, consisted with the results miR-194 was reduced in A549/DDP. (Alterations of microRNAs in cisplatin-resistant human non-small cell lung cancer cells (A549/DDP)).
In summary, we showed that low levels of miR-194 are associated with worse prognosis of NSCLC and could inhibit cell proliferation and invasion in vitro and in vivo. Furthermore, a Together, our findings suggest that miR-194 acts as a tumor-inhibiting factor in NSCLC, and could be a target for NSCLC, even patients with chemotherapy tolerance.

Patients and tissue samples
Paired NSCLC tissue and adjacent normal lung tissue samples were obtained from 64 patients who had undergone surgical resection without prior radiotherapy or chemotherapy between April 2008 and May 2012 at the department of Respiration of Shanghai 10th People's Hospital, affiliated to Tongji University in Shanghai, China. Histopathologic, diagnostic, and clinicopathologic staging of the patients from which the samples were obtained was performed according to the current International Union Against Cancer (UICC) protocol. The use of these tissue specimens was approved by the ethics committee of Shanghai 10th People's Hospital and conducted in adherence with the Declaration of Helsinki protocols. Written informed consent was obtained from all patients. Specimens were frozen in liquid nitrogen and stored at −80°C for further use.

Cell culture
Six NSCLC cell lines (A549, SPC-A1, 95C, 95D, NCI-H1299 and SK-MES-1) and a normal human bronchial epithelial cell line (16HBE) were purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China) and cultured in DMEM supplemented with 10 % fetal bovine serum (FBS) (Invitrogen, Carlsbad, USA), 100 units/ml penicillin and 100 μg/ml streptomycin (Hyclone, Logan, USA) in a humidified incubator at 37 °C with 5 % CO 2 . A cisplatin-resistant human non-small cell lung cancer cell line (A549/DPP) was established and conserved by our lab laboratory and maintained in a1 μg/l final concentration of cisplatin as previously described [9].

RNA extraction and quantitative real-time PCR
Total RNA was extracted from cells using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. For FOXA1 expression, reverse transcription was performed with Superscript first-strand synthesis system (Invitrogen) according to the manufacturer's instruction. The following primers were used: FOXA1 forward, 5′-GC AATACTCGCCTTACGGCT-3′ and reverse, 5′-TACA CACCTTGGTAGTACGCC-3′; GAPDH forward, 5′-ACA ACTTTGGTATCGTGGAAGG-3′ and reverse 5′-GCC ATCACGCCACAGTTTC-3′. GAPDH was used as a loading control. To quantify miR-194, the expression level of miR-194 was measured by the TaqMan miRNA Assay (ABI) according to the manufacturer's protocol. U6 snRNA were used as an internal loading control. qRT-PCR was performed on an ABI 7900HT instrument (Applied Biosystems, USA).All reactions were run in triplicate and all experiments were carried at least three independent times.

Plasmid construction and lentivirus production
An overexpression of miR-194 vector was created according to our previous publications [9] with following primer 5′-AAAGGATCCCAGGAGTTGTAAA TCCGAGCCG-3′ and the reverse primer 5′-AAAGAATTC TTCATAGGTCAGAGCCCTGTGCA-3′. A full length of human FOXA1 3′-UTR with a wide type and mutant target sequence for miR-194 were cloned into downstream of the of Renilla psi-CHECK2 (Promega, Madison, WI, USA) according to our previous publications. To force expression of FOXA1, DNA fragment coding FOXA1 protein was amplified by PCR and then subcloned into the pcDNA3.1 vector (Clontech, Palo Alto, CA, USA) with primers: Forward: 5′-AAAGCTAGCATGTTAGGA ACTGTGAAGATGGAAG-3′; Reverse: 5′-AAACTCGA GCTAGGAAGTGTTTAGGACGGGTCT-3′. All plasmids were confirmed by DNA sequencing.
The lentiviruses of miR-194 were generated by co-transfecting HEK293T with pPACKH1 Lentivector Packaging KIT (SBI) according to our previous publications [9]. To generate the stable cell line, cells were transfected with lentiviruses for 24 h and replaced with complete culture medium. Infection efficiency was confirmed by qRT-PCR 96 h after infection.

Luciferase reporter assay
The luciferase reporter gene vector with wild type FOXA1 (FOXA1-WT) or mutate FOXA1 (FOXA1-MUT) co-transfected with miR-194 in HEK293T cells. Cells were washed 48 h after transfection, lysed and assayed using Dual-Luciferase Reporter Assay reagent (Promega, Madison, USA) according to the manufacturer's instructions.

Cell viability, colony-formation, cell-cycle, and apoptosis assays
Cell viability was evaluated using the Cell Counting Kit 8 (CCK8, Dojindo, Japan) described in our previous publications [9]. In brief, cells were seeded in 96well plates at a density of 1 × 10 3 cells/well in 100-μl medium. 100 μ CCK-8was added to the culture at 1, 2, 3 days. Then the OD value was measured at 450 nm in a spectrophotometer (Spectra Max Plus, Molecular Devices, Sunnyvale, USA).
Cells were seeded into 6-well plates as 800 cells/ well for 14 days. Then, the medium was refreshed with medium containing 10% FBS once every 3 days, and the colony formation was observed. Colonies with more than 40 cells were counted. Four fields were employed, and the mean was obtained. In addition, crys tal violet staining (Shanghai Jingtian Biotech Co., Ltd., JT-00518) was applied and representative photographs were captured.
Apoptotic cells were examined using an Annexin V-PE Apoptosis Detection Kit (Millipore, USA). The samples were harvested and then stained with 5 μl of annexin V-PE and 5 μl of PI. Flow cytometer was performed to detect cellular apoptosis, and the results were analyzed using the Cell-QuestTM Pro software (BD Biosciences, USA). Pretreated cells were harvested, fixed in ice-cold 70 % ethanol at 4 °C overnight, and then resuspended in 1 mL of PBS containing 1 mg/mL RNase (Sigma, St. Louis, USA) and 50 μg/mL PI (Sigma) in a dark box for 30 min at room temperature. The percentage of cells in different phases of the cell cycle was calculated by the Cell-QuestTM Pro software (BD Biosciences).

Migration and invasion assays
The transwell migration and invasion assay was performed using standardized protocols as described earlier [9]. Briefly, 3 × 10 4 cells in 100 μl serum-free media were added to the upper chamber of an insert (8-μm pore size, millepore) coated with or without Matrigel (BD). 600 μl media containing 10% FBS were added to the lower chamber. Cells were fixed and stained with 0.05% crystal violet after 12 hours or 36 hours for the migration and invasion assays, respectively. Six random fields of each chamber were photographed using an IX71 inverted microscope (Olympus, Tokyo, Japan) at 200× magnification.

Western blot
Cells were harvested with 1× cell lysis buffer (Promega). A total of 60 μg of total proteins were separated on 10% polyacrylamide gel and transferred to nitrocellulose membranes (Bio-Rad). The membranes were blocked with 1% bovine serum albumin (BSA) in TBST buffer (Tris Buffer Saline containing 0.1% Tween-20) for 1 h at room temperature, and subsequently incubated with antibodies against FOXA1, BCL-2, CyclinD1 (Proteintech) or GAPDH (#2118, Cell Signaling Technology) overnight at 4°C. After extensive washing with TBST buffer, the blots were then incubated with goat anti-rabbit, horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Protein bands were detected by enhanced chemiluminescence reagents ECL (Millipore, MA, USA) and the intensity of the bands was analyzed using Image J software (National Institute of Health, USA).

Animal model
Six-week-old male nude BALB/c mice were purchased from the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences (Shanghai, China). All mice were housed in specific pathogen-free conditions following the guidelines set forth by the Ethics Committee of the Shanghai Tenth People's Hospital of Tongji University. All protocols were approved by the Animal Care and Use Committee and by the local ethics committee of Tongji University.
To produce experimental tumour formation assay, A549 cells (1 × 10 7 ) infected with lentiviral constructs carrying either the miR-194 vector or the control vector were harvested and were washed and re-suspended in serum-free RPMI-1640 medium, then were injected subcutaneously into the left flanks of anesthetized 4-week-old nude mice. Tumor growth was monitored by measuring the largest (a) and smallest (b) two perpendicular diameters with a caliper, and calculating the tumor volume (V) = a × b 2 × 0.5. Tumor weights were also measured.
To produce experimental metastasis, A549 cells (1 × 10 6 ) infected with lentiviral constructs carrying either the miR-194 vector or the control vector were harvested and injected into the 6-week-old nude mice. Mouse lungs were fixed with 10% neutral-buffered formalin, embedded in paraffin, sectioned at 4 μm and stained with H & E. The number of pulmonary metastatic tumor nodules was counted under a light microscope at magnifications of ×100 and ×200. All the mice were sacrificed at 5 weeks later.

Statistical analysis
Data are presented as the mean ± SD (standard deviation). Student's t-test (two-tailed) or one-way analysis of variance was performed to analyze the in vitro and in vivo data. The association between miR-194 expression and clinicopathological parameters was assessed using the Pearson Chi-Square test. Overall survival was calculated by the Kaplan-Meier method and evaluated by the log-rank test. All statistical analyses were performed using the SPSS version 16.0 software package (SPSS Inc., Chicago, IL, USA). A p-value < 0.05 was considered to be statistically significant.


High Protein for All, is it Currently a State of Art in Critical Care Nutrition?

Proteins and amino acids are widely considered to be subcomponents in nutritional support. However, they are fundamental to recovery and survival, not only for their ability to preserve active tissue (protein) mass but also for a variety of other functions. Although the body adapts in some ways to starvation, metabolic stress in patients causes increased protein turnover and loss of lean body mass.

Introduction
Proteins and amino acids are widely considered to be subcomponents in nutritional support. However, they are fundamental to recovery and survival, not only for their ability to preserve active tissue (protein) mass but also for a variety of other functions. Although the body adapts in some ways to starvation, metabolic stress in patients causes increased protein turnover and loss of lean body mass.

Basis of Protein Requirement
Body protein is in a constant state of protein turnover, with protein synthesis and protein breakdown levels estimated to be in the order of 350g/day. Actually, this is 3.5 -5 times a normal daily protein intake. A reference man of 70 kg contains 10 kg of protein, which is replaced in 28 days.
Herridge et al. [1] followed 109 survivors of ARDS for up to 5 years after discharge from the ICU. All patients reported poor functional status with proximal weakness and fatigue at discharge. At 1 year, the distance walked in 6 minutes was 66 % of predicted which increased to 76 % of predicted at 5 years.

Drawbacks of Protein Deficiency in Critically Illness
The progressive and rapid loss of body mass and muscle is well known to occur in patients with critical illness was termed hypermetabolism. Septic autocannibalism was a term used to describe the loss of muscle mass that does not benefit from increasing AA provision above minimum requirements. The net result is anabolic resistance which is frequently observed in critically ill patients and refers to the state in which a patient is resistant to the normal stimulatory effect of AAs on muscle protein synthesis. Three main factors explain anabolic resistance: splanchnic sequestration of AAs following feeding, which decreases the AAs available to muscles; insulin resistance, which limits AA uptake into muscles and hinders the maintenance of muscle protein; and blunted responses to AAs with anabolic properties, such as the essential AA leucine.

Histological Changes in Skeletal Muscles after ICU Admission
Atrophy of skeletal muscle mass is an almost universal problem in survivors of critical illness and is associated with significant short-and long-term morbidity. The loss of muscle mass is associated with muscle weakness, prolonged mechanical ventilatory support, fatigue and delayed recovery. This disorder is known as critical illness myopathy (CIM) or intensive care unitacquired weakness (ICUAW). CIM is characterized by a diffuse non-necrotizing myopathy accompanied by fiber atrophy, fatty degeneration of muscle fibers and fibrosis. CIM is exceedingly common in ICU survivors, being reported in up to 46 % of cases. Zudin A et al. [2] proved in his study when evaluated critically ill patients with histopathological analysis in addition to B-Mode ultrasound and biochemical analysis that skeletal muscle necrosis and muscle wasting occurred early and rapidly during the first week of critical illness and was more severe among those with multiorgan failure. Interestingly, the incidence of acute myofiber necrosis affected 40% of patients in his study.

Persistent Inflammation, Immunosuppression, Catabolism Syndrome (PICS)
It is a new phenotype of multiple organ failure and a new horizon for surgical ICU which is more common in elderly patients > 65 years. There is a considerable portion of patients that survive acute critical illness and MOF who entered into chronic critical illness (CCI -defined as being in the ICU for > 14 days with ongoing low grade organ dysfunction). Gentile and Moore et al, characterized PICS as: ICU stay >14days, C-reactive protein > 150 Kg/dL, Total lymphocyte count < 0.8, weight loss >

Summary
Protein is an integral part of critical care nutrition. It is needed to build, maintain and repair body tissue and also utilized for energy in increasing amounts during stress (1gm protein provides 4 KCls). Critically ill patient can lose up to 1 kg of LBM daily and loss of LBM is associated with increased mortality. We have suggested earlier that protein intake is not a one size fits all. Using protein intake per kilogram fat-free mass as a reference, we have proposed adjusted body weight for underweight and obese critically ill patients. Protein requirement based on the new ASPEN 2016 should be in a range of 1.2 -2 gm/kg/day which is very difficult to be achieved with current EN formulae, and it has to be supplemented with extra protein added to EN. Surgical subsets and renal failure patients on renal replacement therapy should receive even a higher protein requirement (1.2 -2.5gm/ 10% during hospitalization or body mass index < 18, creatinine height index < 80%, azlbumin level < 3gm/dl, pre-albumin level< 10mg/dl, retinol binding protein < 10mcg/dl. PICS patients were observed to clinically lose tremendous amounts of lean body mass despite optimal nutrition causing profound weakness, suffer from recurrent nosocomial infections, decubitus ulcers, and have poor wound healing.
The persistent smoldering inflammatory and catabolic state, produces a "cachexia" phenotype for which current ICU interventions are ineffective, rendering PICS extremely difficult to treat. Stopping this process, slowing the progression, or even reversing PICS presents many challenges. Currently, there is no existing literature for nutritional support in PICS, but, as defined earlier, one of the main characteristic of these patients is profound malnutrition.

Does Loss of LBM is associated with Mortality?
Loss of LBM is devastating and mortality is approaching 100% in case of loss of 40% of LBM so whatever done, the outcome is very poor and the patient will be at high risk of death (

What is the Effect of High Protein on LBM?
The realistic aim of nutrition support is to blunt the loss of LBM. Protein consumption does three things related to lean mass. First, protein breaks down to amino acids. Amino acids are the building blocks for virtually every tissue in the body, including muscle. In critically ill, because of anabolic resistance, there will be an increase amino acid needs, which is why it's difficult to maintain muscle. Second, protein consumption stimulates protein synthesis. Third, it reduces protein breakdown. When the rate of protein synthesis is greater than protein breakdown, the body produces a net increase in lean body mass. When protein breakdown exceeds synthesis, the body loses lean body mass.

Does Increased Protein Delivery Reduce Mortality in ICU Patients?
Studies agreed on the importance of adequate protein supply ≥1.2 g/kg/day for outcome. As proved in Alberada et al. [3] Every Additional 30gm Protein/day is Associated with Reduced Mortality. Also Van Zanten et al. proved in the MetaPlus randomized, double-blind, multicenter trial that Among adult patients on mechanical ventilation in the ICU, High-protein enteral nutrition enriched with immune-modulating nutrients (IMHP) compared with standard high-protein enteral nutrition (HP) did not improve infectious complications or other clinical end points and may be harmful as suggested by increased adjusted mortality at 6 months. So this trial stressed on the importance of high protein rather than anything else. Allngstrup MJ, et al. [4] showed in his Prospective observational cohort study of 113 ICU patients in a tertiary referral hospital that in these severely ill ICU patients, a higher provision of protein and amino acids was associated with a lower mortality. This was not the case for provision of energy or measured resting energy expenditure or energy or nitrogen balances.

When to Start High Protein Supplements, Early or Late?
Peter JM Weijs et al. revealed that in mixed medical-surgical critically ill patients with prolonged mechanical ventilation (>72 hours), that in non-septic critically ill patients, early high protein intake was associated with lower mortality 37% for <0.8 g/kg, 35% for 0.8 to 1.0 g/kg, 27% for 1.0 to 1.2 g/kg, and 19% for ≥1.2 g/kg (P = 0.033). For these, a protein intake level of ≥1.2 g/kg/day was significantly associated with lower mortality (OR 0.42, 95%CI 0.21 to 0.83, P = 0.013) and early energy overfeeding with higher mortality. In septic patients early high protein intake had no beneficial effect on mortality. PepUp protocol created by Heyland and has been published in Canadian Critical Care Nutrition Website suggested to start early protein (Penny protein 14 gm in 120 ml sterile water tid in addition to high protein semi elemental feed containing peptide, Peptamen) in all critically ill patients including septic or a septic (except hepatic encephalopathy and renal failure patients). Protein lost in critically ill trauma and sepsis patients were observed to be 2 kg in 10 days for the 0.9 g/ kg protein intake level but only half (8%) at 1.2g/kg. Therefore, early protein requirement should at least be considered.

Does the Type of Protein Matter?
Despite, it has been not mention in the ASPEN 2016, Whey protein is considered by experts to be an excellent source of protein. It is one of the two proteins found in milk, with the other being Casein Protein and it is the water-soluble part of milk. Whey is used as a protein supplement and very useful for hitting targeted daily protein goals. It is absorbed faster than other forms of protein, which means it also increases muscle protein synthesis used to break a fasted state [5][6][7][8][9][10][11][12][13][14][15][16][17][18].
Whey-based protein (high in leucine) increases muscle synthesis compared to soya or casein-based protein. Whey protein increased blood levels of essential amino acids and the creation of muscle protein. Arginine enriched enteral formula is recommended in trauma patients especially traumatic brain injury. Glutamine enriched enteral formula, till recently, it has been recommended as a standard of care for high risk surgical, trauma and burn patients but after REDOX trial being negative, the new ASPEN 2016 recommended against its routine use in critically ill patients. Despite the fact that glutamine has been used in these study in a very strange way, e.g. very high dose, both enteral and parenteral and most of the patients was in shock and MOF which is totally Contrary to common practice. Most experts believe that enteral glutamine is still very important for those selective group of patients awaiting new evidence.

Should we Reduce Protein Supplementation in Critically Ill Nephrology Patients?
The ASPEN guidelines suggested the use of standard ICU recommendations for protein (1.2-2 g/kg actual body weight per day). If electrolyte imbalances develop, specialty formulation designed for renal failure maybe considered. They recommend the use protein up to a maximum of 2.5 g/kg/d in patients receiving frequent hemodialysis or CRRT.

What about the Surgical Subsets of Critically Ill Patients?
High protein requirements at range of 1.2-2.5g/kg/day together with 15 -30 grams per liter of volume lost in the negative pressure management systems required in patients with open abdomen. The use of immune-modulating enteral formulas containing arginine and fish oil should be considered in the majority of these surgical subsets. The evidence is lacking in burn population.

Does it Matter Enteral or Parenteral Protein?
There is no randomized control trial looked at the supplemental parenteral protein alone on top of inadequate EN versus inadequate EN alone. So still the ASPEN 2016 is talking about protein and calories as a one package and the recommendation is to start TPN as soon as possible in patient at high nutrition risk (NRS-2002 ≥ 5 or NUTRIC score ≥ 6) or severely malnourished, and when EN is not possible. While patients with low nutrition risk who are unable to meet at least 60% of energy and protein requirements by EN route alone to consider supplemental PN only after 7-10 days.

What is the Current Protein intakes in ICU Patients?
According 2013 survey, the average daily protein given to critically ill patient was only 0.8 gm/kg/day.