Long noncoding RNAs are emerging as key players in various fundamental biological processes. We highlight the varied molecular mechanisms by which lncRNAs modulate gene expression in diverse cellular contexts and their role in early mammalian development in this review. Furthermore, it is being increasingly recognized that altered expression of lncRNAs is specifically associated with tumorigenesis, tumor progression and metastasis. We discuss various lncRNAs implicated in different cancer types with a focus on their clinical applications as potential biomarkers and therapeutic targets in the pathology of diverse cancers. The ‘Central Dogma’ of life describes the flow of genetic information from DNA to proteins involving RNA as an intermediate. The view of DNA being the store house of genetic information and proteins as its functional manifestation dominated the field of biology for several decades. However, the proposal of the famous RNA world hypothesis marked the beginning of an era where RNA was attributed more recognition in terms of cellular and physiological functionality rather than being considered only as a messenger between DNA and proteins . After tIrrespective of the major differences between mRNA and lncRNA with reference to their protein coding capacity, they share some common features as well. Similar to mRNA, most lncRNAs are transcribed by RNAPolII machinery and actively transcribed lncRNA genes possess histone modification signatures similar to that of protein coding genes , 12. TheIt is being increasingly recognized that lncRNAs play a critical role in modulating genetic networks and signal transduction pathways during development and their deregulation leads to disease phenotypes , 20. SevA large number of lncRNAs remain in the nucleus and play an essential role in shaping the epigenome either by genomic imprinting or through chromatin remodeling as described below.Genomic Imprinting refers to the phenomenon of epigenetic silencing of an allele inherited from either of the parents . Short sThe principal means by which most of the lncRNAs regulate gene expression is by recruiting chromatin remodelers to facilitate histone modifications at specific gene loci either for the repression or activation of the target genes . VariousRecent studies have elucidated the fact that several lncRNAs modulate gene expression by specifically associating either at the promoters or the enhancers of their target genes.Divergent transcription at the promoter regions of various genes gives rise to lncRNAs which in turn regulate the transcription of the neighboring genes –52. For Enhancers are critical regulatory elements required for the tight developmental and tissue specific regulation of gene expression and hence it is not surprising that the number of enhancers in mammalian genome far exceeds the number of protein coding genes , 58. VerLncRNAs are also widely implicated in the post transcriptional regulation of mRNAs including splicing, transport, translation and degradation. For example, MALAT1 is involved in splicing events , discussSome lncRNAs regulate gene expression through their interaction with hormone receptors. For example, SRA (Steroid receptor RNA Activator) lncRNA is a coactivator of various steroid hormone receptors like GR (Glucocorticoid Receptor), AR (Androgen Receptor), ER (Estrogen Receptor) and PR (Progesterone Receptor) . GAS5 isWidespread studies have established that lncRNAs participate in a variety of mammalian developmental processes like regulating lineage commitment and cell fate decisions, in organogenesis, in imprinting of alleles during early development and also in specification of the body pattern. A majority of the lncRNAs exhibit a tissue-specific expression pattern , which hi (inactive X chromosome) via H3K27 trimethylation of the chromatin [a (active X-chromosome) [. Like the negative regulator of XIST, there exists a positive regulator, JPX, that in turn is produced from the X-inactivation center and exerts its action in trans to activate XIST on Xi [a-Xi provides a wonderful illustration of RNA-based transcriptional control.An excellent example of genome level regulation has been provided by the discovery of XIST (X inactive-specific transcript). Being involved in inactivating one of the pair of X chromosomes, its expression is restricted mainly to females and further only from the X-chromosome that will be inactivated in the future . Analysihromatin , TSIX isomosome) . Like thST on Xi . This TSXist gene and interestingly discovered that mutant males were unaffected by the deletion along with mutant females who inherited the deleted gene maternally. However, mutant females containing the paternally inherited deletion showed death early during embryogenesis. This was attributed to the expression of both X chromosomes in the extra-embryonic tissues that led to abnormalities in the development of the embryo proper.Loss of function studies for XIST further emphasize its importance in mammalian development. Marahrens et al.  generateHox clusters of genes. LncRNAs have been associated with this phenomenon, a predominant one being HOTAIR [trans mode of action of lncRNAs as it is expressed from the HoxC locus in mammals but exerts its action at the HoxD locus. HOTAIR recruits the PRC2 complex at the target locus resulting in spreading of H3K27 trimethylation over the region and additionally interacts with the LSD1/REST/co-REST complex to perform lysine 4 demethylation, exemplifying the functioning of lncRNAs as molecular ‘scaffolds’ [Hox locus is in fact quite a storehouse of lncRNAs. HOTTIP is expressed at the 5’ end of the HoxA locus and recruits the WDR5/MLL complex across the locus by chromosomal looping, bringing about H3K4 trimethylation and subsequent gene transcription. Interestingly, its strength of action on the Hox genes decreases with increasing distance from its own site of transcription [HoxC locus, FRIGIDAIR has a function in anterior patterning [Hox loci is thus crucial in proper embryonic development.The specification of the anterior-posterior body axis and determination of the positional identity of individual cells as well as organs is governed by a group of homeodomain containing proteins, encoded by the g HOTAIR . It repraffolds’ . The Hoxcription . While Htterning . The comHotair locus has revealed that the lncRNA is as essential as the HOX proteins for the proper development of the embryo [HoxD and certain imprinted loci like Dlk1, Igf2  and H19, Meg3  amongst others. Perturbations in these genes further alters gene expression pattern in vivo leading to abnormalities during development.Targeted deletion at the e embryo . Its absSlc22a2 and Slc22a3, expressed upstream from Air [Kcnq1, Cdkn1c, Phlda2 and Slc22a18) or placental-specific imprinted  [LncRNAs have also been implicated in genomic imprinting of specific alleles, a phenomenon that is a part of the early developmental regime. AIR (Antisense Igf2r RNA) is expressed in an antisense direction from the Igf2r (Insulin-like growth factor type2 receptor) locus, is maternally imprinted and assists in the imprinting of certain paternal genes like from Air . Early dfrom Air . KCNQ1OTd Ascl2) . The lncd Ascl2) .Igf2 locus and itself being paternally imprinted [H19 gene results in mutant animals that are viable and fertile, showing an overweight phenotype probably due to a gain of biallelic expression of the previously imprinted Igf2 locus [Igf2, Slc38a4 and Peg-1 by interacting with the methyl-CpG-binding domain protein, MBD1 [LncRNA H19 is also involved in allelic imprinting, being expressed from the mprinted , 85. It mprinted . Knockouin, MBD1 . The recin, MBD1 .LncRNAs have been well characterized in many cellular contexts and shown to help in maintenance of pluripotency of stem cells, in adult progenitor cell proliferation as well as in the differentiation of specific tissues –93. FurtCancer arises due to accumulation of genetic and epigenetic alterations in cells. Gain or loss of chromosomes has also been frequently observed in cancer cells. Several signal transduction pathways like Wnt/ β Catenin, MAPK, TGFβ, p14ARF/p53, PI3K/ AKT etc. are altered in the malignant cells which seem to produce their own growth factors and attain replicative immortality, enhanced angiogenesis and proliferation. Further, they evade growth repressors, escape apoptosis and acquire the ability of metastasis and invasion . TranscrH19 is among the earliest lncRNAs discovered and identified to be key factor regulating gene expression . ExpressKCNQ1OT1 (KCNQ1 Overlapping Transcript 1) is another imprinted, paternally expressed 91.5 kb transcript produced from the KCNQ1 locus, a few hundred kilobases away from H19 . It reguANRIL (Antisense Noncoding RNA at INK4 Locus), also known as p15AS, is an antisense transcript of CDKN2B gene at the 9p21.3 locus. It has several alternatively spliced isoforms including 3.9 kb and 34.8 kb transcripts , 137. MiXIST , one the earliest lncRNAs to be discovered , is exprInterestingly, XIST is expressed in males also, specifically in the transcriptionally inactive XY body in spermatocytes , though Not only in male and female cancers, XIST is implicated in sex independent cancers as well, mainly in lymphomas and leukemias. Expression of XIST is lost is these cancers resulting in extra active X chromosomes in both male and female patients of non-Hodgkin lymphoma –159. Thutrans silencing of genes at HOXD locus on chromosome 2 [HOTAIR (HOX Transcript Antisense Intergenic RNA) is a 2.2 kb lncRNA produced from the HOXC gene cluster on chromosome 12 (12q13.13) and is involved in the mosome 2 , 77. It mosome 2 , 77.HOTAIR is known to repress several tumor and metastasis suppressor genes like HOXD10 (Homeobox D10), PGR (Progesterone Receptor), PCDH10 (Protocadherin10), PCDHB5 (Protocadherin Beta 5), JAM2 , etc. –162 and While the above discussed lncRNAs are involved in gene regulation at epigenetic level, certain other lncRNAs are involved in transcriptional/ post transcriptional events, as exemplified by NEAT1 and MALAT1, the aberrant expression of which results in cancer.NEAT1 (Nuclear Enriched Abundant Transcript 1) gene produces two transcripts, the 3.7 kb NEAT-1-1 short isoform and 23 kb NEAT-1-2 long isoform. NEAT1 is widely expressed across several tissues, though the expression of long isoform is much lower as compared to the short isoform. NEAT1 localizes to the paraspeckles in the nucleus , 173 andin vitro in urothelial carcinoma [in vivo [MALAT1 (Metastasis Associated Lung Adenocarcinoma Transcript1) is another prominent lncRNA implicated in a variety of cancers. Also known as NEAT2 (Nuclear Enriched Abundant Transcript 2), it is a 7.5 kb RNA transcribed from the 11q13.1 locus, expressed broadly across various normal human tissues , 176. MAarcinoma , 181 and[in vivo .Certain lncRNAs like SRA and GAS5 mediate gene regulation through interaction with hormone receptors and lead to cancer when deregulated, as discussed below.trans activation of genes through its interaction with the AF1 domain of nuclear receptors [SRA (Steroid receptor RNA Activator), an lnc RNA transcribed from the 5q31.3 locus, 2 kb in size, is a coactivator of various steroid hormone receptors as discussed earlier. It has been reported that the SRA1 gene plays a dual role and codes both for a protein (SRAP) and an lncRNA (SRA), by alternative splicing –192. Theeceptors . Its oveeceptors , 194, uteceptors  and proseceptors  cancers.GAS5 (Growth Arrest Specific 5) gene at 1q25.1 locus produces two splice variant lncRNAs and its introns also give rise to several snoRNAs . GAS5 fuTelomeres, the ends of chromosomes, are composed of a hexanucleotide repeat, TTAGGG in vertebrates which protects and prevents end to end fusion in chromosomes . The telTranscribed Ultraconserved Regions (T-UCRs) are evolutionary highly conserved sequences between orthologous regions of the human, rat and mouse genomes , 214. ThWhile the above mentioned lncRNAs are implicated in multiple cancers, certain other lncRNAs have so far been linked only to specific cancer types so far as discussed below.HULC (Highly Up-regulated in Liver Cancer), 1.6 kb in size, is transcribed from the 6p23.3 locus. It was discovered by Panzitt et al.  with theHEIH (High Expression In HCC) is a 1.6 kb oncogenic polyadenylated transcript generated from the 5q34.3 locus. Yang et al.  examinedPCA3  is derived from the 9q21.22 locus, and transcribed as three alternately spliced isoforms of 0.6 kb, 2 kb and 4 kb . It is ePCGEM1 (Prostate Cancer Gene Expression Marker), 1.6 kb in size, derived from the 2q32 locus, it is one of the earliest oncogenic lncRNAs discovered. It is regulated by Androgen Receptor (AR), a transcription factor which has a critical role in the prostate gland development . PCGEM1 PCAT1 (Prostate Cancer Associated ncRNA Transcript 1) is a 7.8 kb lncRNA transcribed from the 8q24.13 locus, up-regulated in both metastatic and high grade localized prostate tumors. Prensner et al.  identifiThese prostate specific lncRNAs are proving to be very useful in the clinic as diagnostic and prognostic markers in Prostate cancer since the traditional markers like PSA have only limited prognostic value .Anti-NOS2A (Anti Nitric Oxide Synthase 2A) is a 1.9 kb intronless polyadenylated lncRNA expressed in meningomas and glioblastomas, transcribed from the NOS2A (17q23.2) locus. It is involved in the negative regulation of NOS2A, which plays an important role in the neuronal differentiation .HOTAIRM1 is a 483 bp transcript generated from the HOXA cluster. It is a regulator of hematopoiesis and its down-regulation results in the inhibition of several HOXA genes required for hematopoiesis. In APL (Acute Promyelocytic Leukemia), differentiation of hematopoietic precursors gets blocked at promyelocytic stage due to chromosomal translocations involving Retinoic Acid Receptor alpha (RAR α) gene. ATRA (All Trans Retinoic Acid) is used to treat this condition and HOTAIRM1 was found to be induced in ATRA mediated differentiation of APL cells , showingDLEU1 and DLEU2 (Deleted in lymphocytic Leukemia 1 and 2) are two lncRNAs produced from the 13q14.3 tumor suppressor locus  which isApart from the individual lncRNAs associated with cancer, several genome wide microarray analyses in recent years have shed light upon hundreds or thousands of lncRNAs that are deregulated in various cancers –244, furin situ hybridization, qPCR, transcriptome profiling etc. [LncRNAs are not only providing us with a new perspective to our understanding of disease mechanisms but also furnishing fresh therapeutic opportunities –247. In ing etc. , which cLncRNAs can be targeted therapeutically by a variety of approaches including RNAi mediated gene silencing, antisense oligonucleotides, plasmid based targeting, through small molecule inhibitors and by gene therapy as discussed below , shRNAs (short hairpin RNAs), and miRNAs. Even though most of the lncRNAs are known to show nuclear localization, various studies have revealed that they can still be targeted by RNAi mediated intervention .The lncRNA HOTAIR is upregulated and serves as a diagnostic and prognostic biomarker for breast, liver, gastro-intestinal, lung and colorectal carcinomas –171, 253in vivo, they would require suitable delivery vehicles like liposomes, nanoparticles or viruses for proper cellular uptake, prevention of their degradation or accumulation in liver. Nevertheless, several RNAi based therapies are in clinical trials [Although RNAi based therapeutic agents are used to target lncRNAs in cell lines quite effortlessly, l trials , 267, thin vivo [Antisense oligonucleotides are short (13–25) single stranded DNA oligonucleotides complementary to RNA of interest. ASOs are modified to avoid degradation by nucleases and in turn they induce RNase H mediated cleavage of their target transcripts. Several ASOs, mainly those targeting mRNAs are already in advanced clinical trials , 269, whin vivo . Notablyin vivo . DespiteThe molecular interactions of lncRNAs with their interacting protein partners can be blocked by small molecule inhibitors that mask the binding sites on their interactors . AccordiIn a novel therapeutic approach, a plasmid, BC-819/ DTAH19, has been developed which carries a diphtheria toxin subunit under the regulation of H19 promoter. When this plasmid is injected into the tumor, it brings about the reduction in tumor size due to the production of high level of diphtheria toxin in human trials of bladder cancer . This meSome lncRNAs are down-regulated in tumor samples as compared to normal tissues. The lncRNA PTCSC3 (Papillary Thyroid Carcinoma Susceptibility Candidate 3) is down-regulated in thyroid tumors . PTENP1,in vivo would be quite challenging and effective strategies need to be developed for the same [in vivo in knockdown/ knockout models in mice. Another point of concern is the fact that even though it has been well established that altered expression of lncRNAs is associated with various cancers, it has not yet been clearly recognized whether the alteration is a cause or consequence of the disease. This calls for a thorough understanding of structure and mechanism of lncRNAs, their molecular interactions and development of novel quantitative assays to screen for drugs. Nonetheless, lncRNAs offer new hope for novel treatment options and in the near future it is expected that many of the lncRNAs may end up as strong diagnostic tools for cancer detection and patient management in the clinic. Because of the increasing number of cancer cases and its incurable nature, there is always a need for novel biomarkers for diagnosis, prognosis and therapy.In summary, though the above discussed means of targeting long noncoding RNAs for cancer therapy looks very promising in cell lines, the delivery of therapeutic agents to their specific targets in actual patients the same . AlthougKey oncogenic and tumor suppressor lncRNAs suggested as potential biomarkers/therapeutic targets are summarized in Table Cancer, being an incurable disease so far, needs novel and effective biomarkers and therapeutic strategies. It is becoming increasingly apparent that deregulated lncRNAs form a new stratum of intricacy in the molecular makeup of human diseases. Their role and mode of action in various signaling pathways during normal and disease conditions is being dissected meticulously and their significance is being acknowledged widely. LncRNAs are strongly associated with clinico-pathological outcome and prognosis of various diseases, more particularly in cancers and furnishing fresh therapeutic possibilities. They are generally expressed in tissue specific manner and exhibit aberrant expression in cancers. Therefore, targeting and either down-regulating or up-regulating specific lncRNAs in malignancies may not have deleterious side effects on normal cells. Thus, of late, both academia and biotech companies are turning their attention towards these novel and possibly personalized treatment options and trying to develop biological/nucleic acid drugs .Various companies/organizations like RaNa Therapeutics, CuRNA, Sarepta, Smart Therapeutics, Allen Institution for Brain Science, Regulus, Miragen Therapeutics, Santaris Pharma etc. are pioneering the ncRNA based medicines. Soon we may see the time when lncRNA signature becomes a routine diagnostic test for diverse diseases, followed up by RNA based therapy curing the hitherto incurable diseases like cancer.


Marburg virus (MARV) was the first filovirus to be identified following an outbreak of viral hemorrhagic fever disease in Marburg, Germany in 1967. Due to several factors inherent to filoviruses, they are considered a potential bioweapon that could be disseminated via an aerosol route. Previous studies demonstrated that MARV virus-like particles (VLPs) containing the glycoprotein (GP), matrix protein VP40 and nucleoprotein (NP) generated using a baculovirus/insect cell expression system could protect macaques from subcutaneous (SQ) challenge with multiple species of marburgviruses. In the current study, the protective efficacy of the MARV VLPs in conjunction with two different adjuvants: QS-21, a saponin derivative, and poly I:C against homologous aerosol challenge was assessed in cynomolgus macaques. Antibody responses against the GP antigen were equivalent in all groups receiving MARV VLPs irrespective of the adjuvant; adjuvant only-vaccinated macaques did not demonstrate appreciable antibody responses. All macaques were subsequently challenged with lethal doses of MARV via aerosol or SQ as a positive control. All MARV VLP-vaccinated macaques survived either aerosol or SQ challenge while animals administered adjuvant only exhibited clinical signs and lesions consistent with MARV disease and were euthanized after meeting the predetermined criteria. Therefore, MARV VLPs induce IgG antibodies recognizing MARV GP and VP40 and protect cynomolgus macaques from an otherwise lethal aerosol exposure with MARV. The Filoviridae family consists of two genera of viruses denoted Ebolavirus and Marburgvirus, which are non-segmented, negative-strand RNA viruses. Filovirus infection of nonhuman primates and humans causes a highly lethal hemorrhagic fever disease with case mortality rates of 30%–90% . MarburgNumerous vaccine platforms have been assessed for efficacy against lethal Marburgvirus exposure over the last 30–40 years . Multipl® and Recombivax HB® hepatitis B vaccines as well as Cervarix® and Gardasil® human papillomavirus-like particle-based vaccines for prevention of cervical cancer [VLPs mimic the particle structure of the authentic native viruses, present the protective viral antigens to the immune system with the correct conformation, but lack a viral genome; thus, these properties engender VLPs to be safe, economical, potent vaccine candidates. In the past several years, VLPs have risen in the vaccine field as a promising vaccine platform for a diverse set of viral pathogens including both nonenveloped and enveloped viruses ,27,28,29l cancer .Expression of the filovirus matrix protein, viral protein 40 (VP40), is sufficient to drive budding of filovirus-like particles . HoweverFor a generation of the MARV VLPs, the MARV GP, NP and VP40 genes were inserted into a single baculovirus vector system for expression in insect cells ,24,38,39in vitro prior to vaccinations, and seronegative for selected retroviruses (simian immunodeficiency virus (SIV), simian retrovirus (SRV) and simian T-cell leukemia virus (STLV)) and filoviruses. Macaques were obtained from Worldwide Primates  and were young adult males (>1.5 years) having body weights of >4 kg to <9 kg.Cynomolgus macaques (Macaca fascicularis) were selected as the test species since they are considered the “gold standard” for testing filovirus vaccines. Inclusion Criteria—to be suitable for this study, NHPs were required to be immunocompetent, testable by polyclonal activation of lymphocytes ad libitum. They were provided toys and treats such as fruit and granola bars. Oral rehydration solution was provided when an animal met the dehydration criteria. Room controls were set to maintain temperatures at 20 to 24 °C (68–75 °F), with a relative humidity of 50% ± 25% with a 12 h light/dark cycle.Animals were singly housed in stainless steel cages and supplied with a standard primate diet (Purina 5L07 diet) throughout the study. Water was available i.e., health checks were performed a minimum of three times per day). For days 22–28 after challenge (study days 134–140), observations returned to once daily, unless the animal was considered ill. The attending veterinary staff provided routine veterinary medical care during the experiments and were on call 24 hours per day to provide emergency care if required.Animal care personnel observed the macaques daily for general health, humane treatment, and husbandry conditions. For 21 days after challenge (study days 112–133), animals were also observed at least twice daily by scientific investigators  Standard Operating Procedures. Blood specimens were collected aseptically from anesthetized nonhuman primates in “Vacutainer” tubes with a 21 gauge 1.5 inch needle via the femoral vein. Blood sample collection was limited to a maximum of 10% of blood volume based on 6.5 mL/kg  during a single draw or a total of 10% over a two-week period.When an animal became clinically ill and met the predetermined criteria for euthanasia, it was first anesthetized and a final blood sample was collected. Subsequently, euthanasia was performed by intravenous or intracardiac administration of a barbiturate overdose.All macaques that survived until study day 140  were considered to have survived the viral infection. These animals were then anesthetized, bled, and euthanized as described above.Partial necropsies were performed on all of the macaques by a board-certified veterinary pathologist. The following tissues from each animal were sampled for histology: axillary lymph node, inguinal lymph node, mandibular lymph node, mesenteric lymph node, liver, spleen, left and right kidney, larynx, thyroid, trachea, lungs, and mediastinal lymph nodes.This animal research was conducted according to the research protocol approved by the USAMRIID Institutional Animal Care and Use Committee. This was in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adhered to the principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, 2011. The facility where this research was conducted is fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International. All challenge studies and necropsies were conducted under maximum containment in an animal biosafety level 4 (BSL-4) facility at USAMRIID.Thirteen macaques received intramuscular injections in the caudal thigh muscle containing 3 mg  of MARV VLPs and either 0.1 mg of QS-21 adjuvant  or 0.5 mg/kg of polyI:C adjuvant . The three control animals received injections of one of the adjuvants only. Immunizations were performed on study days 0, 42, and 84.Pre-challenge blood samples from each macaque were tested by ELISA for serologic responses to filovirus proteins (purified MARV GP amino acid residues 1 to 649 (GPdTM) or VP40) on study days 0, 14, 28, 42, 56, 70, 84 and 105. GPdTM was expressed in 293T cells . ELISA pMARV was grown on Vero cells  and enumerated using standard plaque assay . Adminis2), bicarbonate (HCO3), chloride (CL), glucose (Glu), blood urea nitrogen (BUN), creatinine (Cre), calcium (Ca), albumin (ALB), total protein (TP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total bilirubin (TBIL). Hematological analysis of whole blood was performed as well and the following parameters were captured: White blood cell count (WBC), red blood cell count (RBC), hemoglobin (Hgb), hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (Plt), percentage of lymphocytes (Ly%) and number of lymphocytes (Ly#). Viremia was determined using standard plaque assay [Blood was collected from each animal on the days specified in ue assay .p = 0.6006). Animals vaccinated with MARV VLPs and polyI:C had higher responses to the VP40 antigen than those vaccinated with MARV VLP and QS-21, specifically at the later time points of days 70, 84 and 105 post vaccination (p = 0.0057).Cynomolgus macaques were vaccinated with MARV VLPs  on studyFor the SQ challenge, the results of virus back titration indicated that each macaque received 315 pfu of MARV .t) of experimental atmosphere inhaled (Vt = Vm × length of exposure) by the aerosol concentration (Ce) (“presented dose”pCe × Vt). This equation assumes constant minute volume and constant aerosol concentration over time with complete (100%) respiratory deposition. Aerosol concentration is calculated by: (Csampler × Vsampler)/(Qsampler × tsampled); where Csampler = the titrated concentration of the sampler, Vsampler = the volume of the collection media in the sampler, Qsampler = the flow rate through the sampler, and tsampled = the total time the sample was taken.For aerosol challenge, the results of the back titration indicated that each macaque received between 40 and 135 pfu of MARV  and by a seven-stage cascade impactor .In Groups 1, 2 and 4, the macaques vaccinated with MARV VLPs and challenged via either aerosol or SQ route, there were no animals with visible clinical signs of filovirus infection. The adjuvant only control macaques presented with typical clinical signs of filovirus infection in macaques. Animal 16-P-S, which was vaccinated with polyI:C alone and challenged by the SQ route exhibited severe depression, moderate rash, and no food intake at day 10 post challenge. Both of the macaques that received QS-21 only  exhibited severe depression, widespread rash, and no food intake at day 10 post challenge. These results are shown in Rectal temperatures of the animals were measured during the challenge phase of the study on days 0, 3, 5, 7, 10, 14, 21 and 28 post challenge. In Group 1 , only one macaque  recorded a temperature increase of >2 degrees Fahrenheit above baseline measurement. This fever was only present on day 10 post challenge and subsequently returned to normal for the remainder of the study. Body temperatures in the other animals in this group were normal throughout the study. In Group 2 , animal 7-MQ-A had a fever on days 10, 14, and 21 post challenge, but its temperature had returned to normal by the end of the study. Body temperatures in the other animals in this group were normal throughout the study. None of the three adjuvant only control animals developed an observed fever. However, animal 16-P-S  did record a large decrease in temperature of 8 degrees Fahrenheit on day 10 post challenge and was subsequently euthanized.Hematology parameters were determined in all surviving animals during the challenge phase of the study on days 0, 3, 5, 7, 10, 14, 21 and 28 post challenge. Abnormal results are summarized in Serum clinical chemistry parameters were determined on days 0, 3, 5, 7, 10, 14, 21 and 28 post challenge. A complete list of tests is listed in In contrast to the VLP-vaccinated animals, numerous abnormalities in the serum chemistry parameters were detected in each of the macaques that received adjuvant only. On day 7 after viral challenge, all three macaques had increased levels of ALT  and AST ; increases in these enzymes are indicative of hepatocellular damage. Two of the macaques also had increased ALP levels, which indicate bile stasis, at day 7 post challenge (2× and 4×). On day 10 after challenge and the day of euthanasia, all three of the animals had markedly increased levels of ALT , AST , and ALP . While only one of these macaques had increased levels of CRE at day 7 post challenge (4×), all three animals had increased CRE levels at day 10 post challenge , as well as >2× increase in BUN levels. Increases in CRE and BUN are indicative of compromised kidney function. One animal had decreased levels of glucose at day 7 post challenge (3×), and all three macaques had decreased glucose levels at day 10 post challenge ; hypoglycemia often occurs in animals that are anorexic and/or moribund.Viremia of the challenged macaques was assessed on study days 0, 3, 5, 7, 10, 14, 21 and 28 post challenge. Detectable circulating virus was only present in the three adjuvant only vaccinated animals starting at day 5 post infection, peaking at study day 7 post challenge and leveling out or slightly decreasing on study day 10 post challenge . There wAll three of the adjuvant control monkeys had gross lesions that are typical of those that occur in cynomolgus macaques with lethal MARV infections. These included numerous red cutaneous macules , a tan to yellow friable liver, an enlarged spleen, and one or more enlarged peripheral lymph nodes. Most of the lesions seen histologically in these animals were also characteristic of MARV infection. Splenic lesions consisted of moderate to marked lymphoid depletion with lymphocyte lysis, acute inflammation, and fibrin deposition A. VaryinAll of the macaques that had been vaccinated with VLPs survived the viral challenge and, as expected, none of these animals had gross lesions suggestive of an active MARV infection at the time of necropsy. The only significant gross lesions seen in these monkeys was mild to moderate enlargement of one or more lymph nodes; this was noted in nine of the 13 animals, which included eight of the aerosol-challenged animals (four monkeys each in Group 1 and Group 2) and one of the SQ-challenged animals (Group 4). Histologically, all 13 monkeys had varying degrees of lymphoid hyperplasia in their spleen C and lymEach of these vaccinated survivors also had one or more foci of very mild subacute inflammation in their liver D. The liFoci of chronic interstitial pneumonia and/or pleuritis were present in seven of the 10 vaccinated survivors that had been aerosol-challenged; four of these were from Group 1 and three were from Group 2. These lesions were probably induced by the aerosol challenge; however, there was no evidence of an active viral infection at the time these animals were euthanized. In five of these seven macaques, the pulmonary lesions were very mild and clinically insignificant. In the other two animals (one from Group 1 and one from Group 2), the pneumonia was more extensive and may have caused clinical signs at some time but it appeared to be resolving and was unlikely to have been clinically significant at the time the animals were euthanized. None of these animals were noted to have clinical signs of respiratory disease during the course of the study; it is also possible that the lung lesions in these monkeys were present before the study began and/or were unrelated to the viral challenge.There were no other remarkable findings in the survivors.In this study, cohorts of cynomolgus macaques were vaccinated with VLPs expressing proteins from MARV (Musoke isolate) in the presence of QS-21 or polyI:C adjuvants or were sham-vaccinated with adjuvant alone. Animals were then challenged with the homologous MARV-Musoke strain either by aerosol or SQ route. The outcome indicates that vaccination with MARV VLP with either adjuvant (QS-21 or polyI:C) provides complete protection against challenge with aerosolized MARV-Musoke. Additionally, as expected, MARV VLP with QS-21 adjuvant provided 100% protection against SQ homologous challenge.This is the first report to date of a subunit vaccine protecting macaques against lethal aerosol infection with MARV. To our knowledge, there is only one other report of a vaccine protecting against aerosolized MARV infection where a multivalent live, replicating vesicular stomatitis virus-based vaccine protected against infection with both Ebola and MARV infections . AdditioIn this study, we further broadened the use of novel adjuvants given in conjunction with the filovirus VLPs in nonhuman primates. We had previously shown that Ebola VLPs administered in combination with RIBI or QS-21 adjuvant provide protection of nonhuman primates against homologous and heterologous parenteral infection  to broadly protect against the viruses known to be virulent in humans. The inclusion of a component for protection against MARV and RAVV should not be dismissed in future efforts for filovirus vaccines. Disease caused by MARV is just as deadly and spread with the same mechanism as Ebola, and there have been a larger number of MARV outbreaks in the last decade than those caused by EBOV . FortunaVaccination of cynomolgus macaques with MARV VLP with either QS-21 or polyI:C adjuvant provided complete protection against challenge with aerosolized MARV-Musoke.


In several genus evolutionary chromosome change involves variation in DNA amount in diploids and genome downsizing in polyploids. The constancy of bimodal karyotypes, even with changes in ploidy level and DNA content per basic genome indicate that the distribution of DNA within the complement is not at random and suggest the presence of mechanisms selecting for constancy, or against changes, in the karyotype morphology. Hippeastrum species with different ploidy level were analysed. In Hippeastrum, polyploid species show less DNA content per basic genome than diploid species. The rate of variation is lower at higher ploidy levels. All the species have a basic number x = 11 and bimodal karyotypes. The basic karyotypes consist of four short metacentric chromosomes and seven large chromosomes (submetacentric and subtelocentric). The bimodal karyotype is preserved maintaining the relative proportions of members of the haploid chromosome set, even in the presence of genome downsizing. The constancy of the karyotype is maintained because changes in DNA amount are proportional to the length of the whole-chromosome complement and vary independently in the long and short sets of chromosomes. This karyotype constancy in taxa of Hippeastrum with different genome size and ploidy level indicates that the distribution of extra DNA within the complement is not at random and suggests the presence of mechanisms selecting for constancy, or against changes, in karyotype morphology.Evolutionary chromosome change involves significant variation in DNA amount in diploids and genome downsizing in polyploids. Genome size and karyotype parameters of  The diversity of plant genomes is manifested through a wide range of chromosome number and genome size . The parVicia (Fabaceae) , Asparagaceae  and Amaryllidaceae   . TaxonomHippeastrum Herb. is a genus of perennial and bulbous plants of the tribe Hippeastreae of Amaryllidaceae J.St.-Hil.  chromosomes and seven large chromosomes (four submetacentric—sm and three subtelocentric—st) (tric—st) . This geHippeastrum diploid species from South America, karyotypes similar to that previously described but significant differences in nuclear DNA content. These authors report that karyotype constancy is a product of changes in DNA content occurring in the whole-chromosome complement, and that DNA addition to the long and short sets of chromosomes varies independently. The authors state that the evolutionary changes in DNA amount are proportional to chromosome length, maintaining karyotype uniformity. They found that in diploid species with higher DNA content, the short chromosomes add equal DNA amounts to both arms, maintaining their metacentric morphology, whereas the long chromosomes add DNA only to the short arm, increasing chromosome symmetry.x to 7x) in several species of the genus (Hippeastrum has not yet been reported. It has been frequently documented that the major trend in vascular plants is a decrease in the genome size per haploid genome (1Cx), when a polyploidization event occurs . The sources of the materials are listed in Table Cytological studies were carried out on material cultivated at the Royal Botanic Gardens, Kew, with the exception of one specimen of iker ATH 8258. TheS and CIL), was calculated according to CL) and the mean centromeric asymmetry (MCA) were calculated according to Hippeastrum and related genera. Chromosomal parameters were measured using the freeware program MicroMeasure 3.3 (http://www.colostate.edu/Depts/Biology/MicroMeasure/). Mean values for the karyotype parameters were measured from a minimum of five scattered metaphase plates in each accession.For squashing, root tips were pretreated for 2.5 h in 0.002 M 8-hydroxyquinoline at 20 °C, fixed in 3 : 1 absolute ethanol : acetic acid and stained in Feulgen solution. The average of centromeric indices, for small and large chromosomes  using generalized linear mixed models. The mean values of genome sizes were calculated and multiple contrasts were performed with the LSD Fisher method . These sThe relationship between total DNA content and ploidy level was studied by fitting a weighted least-squares linear regression. This method compensates for the variable number of DNA measurements available for each species and ploidy level .The statistical analyses were performed using the Infostat program, FCA, National University of Córdoba .Total genome size 2C), DNA per basic genome (1Cx), karyotype formulae and karyotype parameters for diploid and polyploid species are listed in Table C, DNA pex = 11 (Table x = 11) are given in a diagrammatic form  is similar in all the taxa analysed (23.05–25.12) (Table S) are very similar among diploid and polyploid taxa (42.42–46.87). On the other hand, the centromeric indices of large chromosomes (ICL) decrease at lower genome size in diploids (19.9–26.17), while 3x, 4x and 5x present similar values (23.18–24.37). The hexaploid differs from the rest of the species in their karyotype parameters, having a similar CIS but a higher CIL (Table CA and CVCL are given in Table Hippeastrum puniceum (6x), with the lowest basic DNA amount (1Cx), occupies an isolated position when compared with the rest of the Hippeastrum species. This is a consequence of its more symmetrical karyotype.All the diploid and polyploid species presented 11 Table . The relorm Fig. . The volnalysed 2.05–25.12F = 427.44, P < 0.0001). They are indicated in Table y = 8.9x + 13.6; x = ploidy level, R2 = 95 %) but the calculated regression line has a gentler slope than the line extrapolated from the diploid mean, which assumes that when the number of genomes increases DNA is added as an exact multiple of the DNA content per basic genome   varies from 26.80 to 34.17 pg among diploids and increases with ploidy level, reaching a value of 64.67 pg in the hexaploid species. This genus has large genomes, since according to Hippeastrum the polyploids studied show less DNA content per basic genome than diploids. Considering the average of basic DNA content for diploids, the triploid diminishes by 16.77 % while the decrease among 3x–4x, 4x–5x and 5x–6x ploidy levels is lower, varying between 5.5 and 6.5 %. These results show that in Hippeastrum, DNA per haploid genome decreases in polyploids, the rate of variation being lower at higher ploidy levels. Many examples are found in the literature where polyploidy is associated with decreasing genome size, in terms of DNA content per haploid genome. Moreover, comparative genome studies have shown that the downsizing of the genome can take place even in a few generations and could be involved in the genetic and cytogenetic diploidization (Larrea (Zygophyllaceae) (Aloe (Xanthorrhoeacae) (DNA per basic genome (1Cx), calculated from total DNA content, varies from 17.08 to 13.40 pg in diploids. The difference between these extreme values is significant. In polyploids there is a gradual decrease in the 1Cx value when ploidy level increases, varying from 12.90 pg in triploids to 10.78 pg in hexaploids. In dization . While pdization . To explhoeacae) , differeHippeastrum here studied presented x = 11 and despite possessing significant differences in their genome size, all have a basic bimodal karyotype with four small m and seven large sm/t chromosomes. The constancy of the karyotype in taxa of Hippeastrum with different genome size and ploidy level indicates that the distribution of extra DNA within the complement is not at random and suggests the presence of mechanisms selecting for constancy, or against changes, in karyotype morphology, processes named by CA and CVCL to estimate the intrachromosomal and interchromosomal asymmetries, respectively  is very similar, indicating that the volume of long and short chromosomes remains in a similar proportion among species. As previously discussed, this karyotype uniformity occurs if changes are proportional to the relative length of each chromosome arm , joined to the presence of conserved bimodal karyotypes, even with changes in ploidy level and 1Cx value, strongly suggests that DNA changes could occur by amplification or deletion of retroelement sequences, which are generally dispersed in the genome.Hippeastrum, evolutionary chromosome change involves variation in DNA amount in diploids and genome downsizing in polyploids. Besides, the bimodal karyotype is preserved maintaining the relative proportions of members of the haploid chromosome set by karyotype orthoselection. The presence of conserved karyotypes, even with changes in ploidy level and DNA content per basic genome, is strongly susceptible to an adaptive interpretation, suggesting the existence of mechanisms that select for constancy in karyotype morphology.In the genus Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET-PIP 00342), Universidad de Buenos Aires (UBACYT 20020100100859) and Agencia Nacional de Producción Científica y Tecnológica—SECyT (PICT 2010-1665).Funding was provided by grants from the All authors contributed to the experimental design, data analysis and manuscript preparation.None declared.


The present study evaluated the influence of hyperandrogenism on oocyte quality using a murine PCOS model induced by dehydroepiandrosterone (DHEA) and further explored the effect of metformin treatment. Female BALB/c mice were treated with a vehicle control or DHEA (6 mg /100 g body weight) or DHEA plus metformin (50 mg /100 g body weight) for 20 consecutive days. DHEA-induced mice resembled some characters of human PCOS, such as irregular sexual cycles and polycystic ovaries. After the model validation was completed, metaphase II (MII) oocytes were retrieved and subsequent analyses of oocyte quality were performed. DHEA-treated mice yielded fewer MII oocytes, which displayed decreased mtDNA copy number, ATP content, inner mitochondrial membrane potential, excessive oxidative stress and impaired embryo development competence compared with those in control mice. Metformin treatment partially attenuated those damages, as evidenced by the increased fertilization and blastocyst rate, ATP content, GSH concentration and GSH/GSSG ratio, and decreased reactive oxygen species levels. No significant difference in normal spindle assembly was observed among the three groups. During in vitro maturation (IVM), the periods of germinal vesicle breakdown (GVBD) and the first polar body (PB1) extrusion were extended and the maturation rate of GVBD oocytes was decreased in DHEA mice compared with controls. Metformin treatment decreased the time elapsed of GVBD while had no effect on PB1 extrusion. These results indicated that excessive androgen is detrimental to oocyte quality while metformin treatment is, directly or indirectly, beneficial for oocyte quality improvement. Polycystic ovary syndrome (PCOS) is the most common female endocrine disorder and has a cluster of characteristics, such as oligo-ovulation or anovulation, hyperandrogenism and multicystic ovaries. Metabolic abnormalities, including obesity, insulin resistance, type 2 diabetes and cardiovascular disease are also found in PCOS. Further, PCOS is one of the main reasons for female infertility, affecting 5–10% women of reproductive age. During in vitro fertilization (IVF) treatment, although PCOS patients obtain increased oocytes, they often have lower rates of fertilization, cleavage and implantation and a higher rate of miscarriage, which might be associated with poor quality of the oocytes and the resulting embryos,4, suggeIn addition to the fecundity effect, hyperandrogenism can also prompt insulin resistance and induce metabolic disturbances in PCOS patients ,6. MetfoRecently, a study by Palomba S et al. demonstrated that metformin reduced the intrafollicular androgen level and improved ovarian insulin resistance and ovary morphology in PCOS patients, suggesting a local benefit on the ovary. In addiAll of the chemicals used in this study were purchased from Sigma-Aldrich Chemical Co.  unless otherwise stated.All procedures involving mice were operated under strict criteria on the basis of the Guide for Care and Use of Laboratory Animals of Peking University, and the protocol was approved by the Institutional Animal Care and Use Committee of Peking University Third Hospital.in vitro fertilization and in vitro maturation.Female prepuberal (25 days old) mice of the BALB/c strain  were randomly divided into three groups . Animals of the DHEA group were injected daily with DHEA  and given saline (0.2 ml) orally with a cannula for 20 consecutive days. The DHEA+metformin group animals were injected daily with DHEA and given 1,1-dimethylbiguanide hydrochloride  orally for 20 days. The control group animals were administered oil (0.05 ml) and saline (0.2 ml). All of these mice were raised and housed in the Animal Center of the Medical College of Peking University according to the national legislation for animal care. All mice were maintained under controlled temperature and lighting conditions and allowed free access to food and water. Throughout the whole treatment period, the animals were weighed every two days, and vaginal smears were taken daily beginning 10 days after the first injection up to the end of experiments. The stage of cyclicity was determined by microscopic analysis of the predominant cell type in vaginal smears. After 20 days of treatment, eight to ten mice per group were assessed whether the model was successfully established. As all mice from DHEA and DHEA+metformin group were completely acyclic and remained in constant estrus while control mice had normal cycle. To eliminate the effect of estrous cycle on other detecting indicators, only those mice that sacrificed on the estrus stage in the control group were used for model validation. Magnetic resonance imaging (MRI) and oral glucose tolerance tests (OGTT) were performed before the mice were anaesthetized with ether and euthanized by cervical dislocation. The rest of the animals were used for oocyte collection and the following quality evaluation. 7–8 weeks old mice were used in all experiments, with the exception of the body weight and the estrous cycle evaluation. During oocyte quality evaluation, 3–5mice per group was used in each experiment except for 6–10 mice per group was used in To determine body fat mass composition, mice were placed in a clear plastic holder without anesthesia or sedation and inserted into the EchoMRI TM device  at the end of the treatment.Mice were fasted for 8 hours before the OGTT experiment. Glucose levels were measured by tail vein blood sampling using a blood glucose meter . After the fasting, the glucose levels were measured, then the mice were administered glucose (2 g/kg body weight) by oral gavage, and tail sampling was performed at the time point of 30, 60, 90and 120 minutes.At the end of the treatment, blood was drawn from the inner canthus after the mice were fasted for 8 hours. The serum was immediately separated and kept in -80°C for subsequent hormone determinations. Testosterone and fasting insulin levels were measured with radioimmunoassay kits . Lipid profiles were determined with biochemical analysis kits .Immediately after the collection of blood samples, the ovaries were quickly removed and fixed in 4% paraformaldehyde. After embedding in paraffin and following routine histological procedures, 5-μm sections were mounted on glass slides and stained with hematoxylin and eosin for observation under the light microscopy.To collect MII oocytes, mice from the control, DHEA and DHEA+metformin groups were superovulated by the intraperitoneal injection of 10 IU of equine chronic gonadotropin , followed by 10 IU of human chorionic gonadotropin  48 h later. Cumulus-oocyte complexes (COCs) were collected from oviductal ampullae 14–16 h after hCG injection, and the cumulus cells were removed by brief incubation in 0.2% hyaluronidase. Denuded oocytes were observed under stereomicroscopy. Oocytes with a round clear zona pellucida, a small perivitelline space and a pale, moderately granular cytoplasm that does not contain inclusions were considered to be “normal” , and onlTo collect GV oocytes, ovaries were obtained from mice that were not stimulated by any exogenous hormone. The collected ovaries were dissected by manual rupturing in G-MOPS , and the separated GV oocytes were collected for in vitro maturation (IVM).2 and 95% humidity. Ovulated oocytes with intact cumulus masses were recovered from oviducts and maintained in HTF. Then, the capacitated sperm in a concentration of 1–2×106/ml was added to the COCs droplet and cultured at 37°C, 5% CO2 and 95% humidity. Six hours later, the oocytes were removed and examined for fertilization. Fertilization was assessed by the presence of two pronuclei (2PN). Twenty to thirty fertilized embryos were transferred to one KSOM (K Simplex Optimization medium) droplet with a 30ul volume at 37°C, 5% CO2 and 95% humidity. Fertilized embryos were cultured to the blastocyst stage for 4 days, and the development was recorded every 24 h.The epididymal spermatozoa were retrieved from the cauda epididymis of 8–10 week-old BALB/c male mice, and the sperm suspensions were capacitated in human tubal fluid (HTF)/10% serum protein substitute (SPS) medium for 90 min at 37°C in 5% CO2 in a live cell station . Twenty denuded GV oocytes were cultured in a 20-μl drop of IVM medium, and pictures were taken every 30 seconds. The beginning time and the time of germinal vesicle breakdown (GVBD) and first polar body (PB1) extrusion were recorded, and the time elapsed until GVBD and PB1 extrusion was calculated. After 24 hours, mature oocytes with PB1 extrusion were collected for spindle and chromosome staining.Immature oocytes were cultured in an IVM medium  for 24h in a humid atmosphere at 37°C with 5% COFor spindle and chromosome analysis, denuded MII oocytes were placed in 4% paraformaldehyde for 30 min after rinsing twice in PBS. Then the oocytes were permeabilized with 1% Triton X-100 and incubated overnight at 4°C. The next day, the permeabilized oocytes were incubated with PBS supplemented with 1% bovine serum albumin (BSA) for 1–2 hours at room temperature. Subsequently, the oocytes were transferred to a droplet containing monoclonal anti-alpha-tubulin-FITC antibody (1:100 dilution in BSA) for 1–2 hours. After three washes, the oocytes were stained with 10 μg/ml of propidium iodide for 5–10 min. Finally, the oocytes were mounted on glass slides and examined with a confocal laser scanning microscope .For inner mitochondrial membrane potential or reactive oxygen species (ROS) staining, denuded MII oocytes were incubated in PBS containing 2 μM JC-1 fluorochrome  or 10 μM DCFH-DA at 37°C, 5% CO2 in air for 30 min. Meanwhile, the negative controls were cultured in PBS for 30min. After washing 3 times with PBS supplemented with BSA, oocytes were placed in glass bottom cell culture dishes  and analyzed by fluorescence microscopy. The fluorescence intensity was measured with Image J software.For lipid droplet staining, denuded MII oocytes were placed in 4% paraformaldehyde for 45 min, washed three times with PBS and then stained with 1 μg/ml BODIPY 493/503 (Invitrogen) lipophilic dye for 1 h in the dark at room temperature. Meanwhile, the negative control oocyte was cultured in PBS for 1 h. After staining, oocytes were washed 3 times in PBS supplemented with BSA and placed on a confocal dish. Images were taken by confocal microscopy at × 20 magnification.For reduced glutathione(GSH) and oxidized disulfide (GSSG) content evaluation, a commercial assay kit  was used. Procedures were performed according to the manufacturer’s instructions and a previous literature . A totalFor the measurement of the ATP content in MII oocytes, a commercial ATP assay kit  was used and the detection was performed with a luminometer . 20–40MII oocytes from each group were vortexed in 100 μl schizolysis solution for 1 minute for lysis. After that, the mixture was centrifuged at 12,000 g for 10 min at 4°C, and the supernatant was collected and stored at -80°C until further use. Eight standard points  were performed in each assay, with the formula derived from the standard curve, the ATP content was calculated.One single MII oocyte was added to a PCR tube containing 10 μl lysis buffer . Then the tube was incubated at 55°C for 2 h and 95°C for 10 min. After that, the samples were used for real-time qPCR detection directly by an ABI 7500 real-time PCR system . The procedure to obtain the purified plasmid standard DNA used to produce a standard curve and the mitochondrial DNA (mtDNA) quantitative real-time PCR were performed as previously described. All of Data are represented as the mean± SEM from at least three replicates per experiment. Statistical significance was determined as indicated; by one-way analysis of variance followed by Bonferroni, Dunnett or Scheffe post hoc analysis, chi square tests, as appropriate using SPSS 16.0 software. A P value of<0.05 was considered statistically significant.To evaluate the reproductive function of the mice, estrous cycle and ovarian morphology were assessed. All control mice had a normal estrous cycle of 4–5 days. However, mice treated with DHEA were completely acyclic and remained in constant estrus. Ovaries from control mice exhibited follicles in different stages of development. Neither structural abnormalities nor ovarian cysts were found in these ovaries . HoweverAt the end of the model establishment, the weight gain and fat mass of the DHEA-treated mice were significantly higher than those of the age-matched controls. However, when metformin was administered simultaneously, body weight and fat mass were significantly decreased , Fig 2. To investigate the influence of DHEA on ovulatory function during superovulation, mice were primed with PMSG and hCG. As shown in To confirm the effects of DHEA and metformin on the development competence of oocytes, the development of fertilized embryos were analyzed. Compared with controls, DHEA treatment significantly decreased the fertilization ability of oocytes and the 2-cell, 8-cell and blastocyst formation rates. As shown in As maternal lipid metabolism was greatly changed by DHEA, we wondered whether the lipid content of oocytes was also influenced. MII oocytes from DHEA mice showed visibly higher levels of lipid than those from the controls (1.62±0.099 vs. 1.01±0.044), whereas metformin treatment (1.11±0.056) completely reversed the lipid augmentation in DHEA mice, which showed the similar lipid content to the controls .The mitochondrion is the primary organelle of metabolism in cells. To evaluate mitochondrial function in MII oocytes, the mtDNA copy number and ATP content were assessed. The average mtDNA copy number  and ATP The mitochondrial membrane potential (MMP) of oocytes was determined by staining the cells with inner membrane potential dye JC-1. JC-1 emits green fluorescence when the MMP Δψm is lower than 100mV and emits red fluorescence when the MMP is increased. The reduction of MMP, determined by the ratio of red to green fluorescence, is considered to be an initial manifestation and hallmark of mitochondrial damage. We founIntracellular ROS was measured to evaluate the oxidative stress in oocytes. The DCFH-DA fluorescence intensity was significantly higher in the DHEA group than that in the control group, suggesting an increased production of ROS. The augmented ROS production was greatly reversed by metformin treatment. Representative images are shown in Fig GSH is an important antioxidant. The GSH level and the ratio of GSH to GSSG are regarded as important parameters of the antioxidant capacity of the body. As shown in Spindle and chromosome assembly of the oocytes matured in vivo was examined by immunofluorescence staining and observed under confocal microscopy. A total of 154 MII oocytes were collected for analysis. As shown in To evaluate the maturation potential of GV oocytes, immature oocytes from the three groups were cultured in IVM medium for 24 h under the live cell station. As shown in Fig In the present study, we investigated the effects of DHEA and metformin on maternal metabolism and oocyte quality in mice. The results indicated that excessive DHEA disrupted maternal metabolism and fertility function, hampered the oocyte quality and decreased in vitro maturation and developmental potential. However, metformin treatment partially rescued these impairments and improved the oocyte quality.In 1962, the observation of increased DHEA levels in PCOS women . Since tPCOS patients are often associated with poor oocytes quality during IVF, and hyperandrogenism might be a main reason. The full developmental competence of an oocyte requires the synchronous maturation of the nucleus and cytoplasm . Any dysAmong the changes that occur during cytoplasmic maturation, the functional status of mitochondria, which are the most abundant organelles in mammalian oocytes, play a primary role. DHEA, an adrenal steroid, is known to have an anti-proliferative effect and is associated with mitochondrial function impairment. Previous studies have shown that DHEA treatment can cause neuronal mitochondrial dysfunction and lipid peroxidation in rat liver mitochondria ,32. In aAlthough the systemic effect of metformin has been identified in PCOS patients, the local effect on oocyte maturation is not quite clear. During the process of maturation, the oocyte obtains most of the energy from cumulus cells, which metabolize glucose via glycolysis to produce pyruvate and lactate, the preferred substrates of the oocyte. MetformHowever, it is worth noting that some previous studies demonstrated that metformin can inhibit the mitochondrial ETC complex I activity and impair mitochondrial function ,45. The As the technology of live cell stations and time lapse has just started emerging, studies of the nuclear dynamics during oocyte maturation have been limited at this point. Escrich’s study demonstrated that the time required for nuclear maturation, which is important for the resumption of meiosis and normal activation, depends on the duration of the GV stage rather than that of MI arrest (time elapsed between GVBD and PB1 extrusion). These rThis study is the first to confirm a direct impairment of excessive androgen on oocyte quality, which can be partially rescued by metformin treatment. However, to achieve the goal of obtaining a PCOS-like mouse model, the DHEA was given in a quite high dosage, which far exceed the androgen levels in PCOS patients, makes it difficult to extrapolate to humans. Also, because of the physiological differences between mice and humans, the mouse model used in this study cannot fully represent the ture situation of PCOS patients. Nevertheless, our study provided a clue that hyperandrogenism might be a direct cause of poor oocyte quality in PCOS patients during IVF, whereas metformin pretreatment might reverse the impairment. Further studies should be conducted to confirm this effect in clinical studies and elucidate the mechanism of the improvement induced by metformin.


Ocular films complied with all physicochemical parameters underwent in-vitro release study. Finally, the film formulation with HPMC: Eudragit RS PO 1:1 ratio, Drug: Polymer ratio 1:45 and glycerin as plasticizer showed controlled and prolonged release following the zero order and non-Fickian transport.Diclofenac sodium as ophthalmic dosage form is used for the treatment of the pain, swelling and redness of patients’ eyes recovering from cataract surgery; however, it faces the bioavailability limitation of eye drops due to effective protective mechanisms and corneal barrier functions in the eyes. Therefore, this investigation was aimed to develop ocular film formulations to achieve controlled drug release. Drug films were prepared using polymers, namely hydroxypropyl methylcellulose (HPMC) and polyvinyl pyrrolidone (PVP), Eudragit RL PO, and Eudragit RS PO by solvent casting method considering parameters such as drug: polymer ratio, different polymer combinations as well as plasticizer effect. Ocular films were evaluated for various physicochemical parameters such as physical characters, film thickness, uniformity of weight, drug content, swelling index, mucoadhesion time and  Due to the poor drug bioavailability, ocular drug delivery still faces many challenges which can be the result of ocular anatomy, physiology and efficient protective mechanism including extensive nasolachrymal drainage, tear dynamics, relative impermeability of the corneal epithelial membrane and the high efficiency of the blood–ocular barrier. The majority of ophthalmic dosage forms administrated in the form of highly concentrated eye drops suffer from short precorneal residence time of eye drops which is associated with low corneal drug absorption and also ocular and systemic side effects. Furethermore, in order to achieve therapeutic effect, frequent administration of these concentrated solutions is required which results in short residence of high drug concentration in the tear film followed by long periods of underdosing ,2. ThereDiclofenac sodium, a nonsteroidal anti-inflammatory drug which is a derivative of phenyl acetic acid, is applied topically as a 1 mg/mL aqueous solution in the eye to manage pain in corneal epithelial defects in surgery or accidental trauma and symptomatic relief of seasonal allergic conjunctivitis. It can also be used to prevent intra-operative miosis during cataract surgery, and also to treat postoperative chronic inflammation and non-infectious ocular inflammation , 12. As mentioned, most of the dose applied topically in the eye from such solutions is lost due to pre-corneal losses. In the literature, there are studies done by pharmaceutical scientists on diclofenac such as liposomes , nanosusin-vitro release study were evaluated.The present study was aimed to prepare diclofenac sodium ocular films with the target of increasing the contact time and offering a controlled release pattern which further could improve patient compliance, reduce the frequency of administration, and obtain greater therapeutic efficacy. Therefore, diclofenac sodium ocular film utilizing various HPMC and Eudargit polymers was prepared and physicochemical parameters such as film thickness, uniformity of weight, drug content, swelling index, mucoadhesion time and Materials Diclofenac Sodium was obtained from Daroupakhsh co., Iran. Hydroxypropylmethylcellulose (HPMC) 4000 cps was purchased from Seppic, France. HPMC 15000 cps and HPMC 100000 cps were purchased from Shandong Co., China. Eudragit RL PO and Eudragit RS PO were obtained from Rohm Pharma, Germany. Polyvinyl pyrolidone K30 (PVP K30) and sodium carboxy methylcellulose (Na CMC) were obtained from Blanver Co., Brazil. Polyethylene glycol 400 (PEG 400), Glycerin and Triethyl citrate (TEC) and all other reagents and solvents of analytical grade were purchased from Merck Co., Germany.Ocular diclofenac sodium film preparation2) and covered with an inverted funnel to allow slow and uniform evaporation at room temperature for 48h. The dried films were cut into pieces of definite size (1 cm2) containing 600 µg/1cm2. Diclofenac sodium ocular films were prepared by solvent casting evaporation technique. The composition of prepared polymeric ocular films has been summarized in Characterization of prepared ocular filmsPhysical characterizationThe physical characteristics of ocular films such as color, texture, flexibility and appearance were evaluated. Uniformity of weightFrom each batch, 3 films were weighed individually using digital balance . The mean weight of the films was recorded. Uniformity of thicknessThe thickness of films was determined using a Vernier caliper . For each formulation, the thickness of 3 randomly selected films was tested .Drug content deteminationOcular films (3 samples) were taken from each batch and dissolved using 50 mL of isotonic phosphate buffer pH 7.4 (tear fluid) into volumetric flask. The absorbance of solution after filteration and required dilution was measured by UV-VIS spectrophotometer  at 283 nm. The mean drug content of films was determined considering the concentartion of the solution and the number of films dissolved.Swelling index testIn order to measure the bulk hydrophilicity and hydration of films, swelling test was done as drug release from the polymeric matrix is affected by swelling. To test the swelling of diclofenac sodium films, three films of each formulation were weighed and put in a mesh basket and inserted into phosphate buffer saline (PBS) of pH 7.4 maintained at temperature of 32±0.5 °C. At time intervals of up to 90 min, the films were removed, wiped with lint-free tissue to remove excess surface PBS, weighed, and then returned back to the same container . The swelling index was calculated using the following equation based on degree of fluid uptake: W0 is the initial weight of the sample and Wt is its weight at time t. Where In-vitro drug release studyin-vitro drug release from different ocular films was studied using the vial method. Each film was placed in a vial containing 10 mL of simulated tear fluid (pH 7.4) which was previously warmed at 37 ± 1°C. These vials were positioned over a Kottermann 4020 shaker. To simulate the eye blinking, the shaker was kept at its minimum shaking speed. Aliquot of samples at specific time intervals was withdrawn and the equivalent amount of fresh fluid was replaced. The samples were analyzed at 283 nm using UV Spectrophotometer after appropriate dilutions against reference using isotonic phosphate buffer pH 7.4 as blank.The Drug release kineticsin-vitro drug release results were fitted with different kinetic models such as zero order (% release vs. t), first order (log % release vs. t), Higuchi matrix (% release vs. t0.5) to understand the kinetics and mechanism of drug release. Data of drug release was further analyzed by Peppas equation, Mt/M∞ = ktn, where Mt is the released drug amount at time t and M∞ is the released amount at time ∞, the Mt/M∞ is the released drug fraction at time t, k is the kinetic constant and n is the diffusional exponent, a measure of the primary mechanism of drug release. The plots of above models were analysed by regression analysis and the regression coefficient (r2) values were calculated for the linear curves obtained  by application of ocular films on a freshly cut sheep eyelid. Ocular film was attached to the mucosal surface of the eyelid fixed on the bottom of a beaker by applying a light force with a fingertip for 20 s. The beaker was filled with 100 mL of bicarbonate Ringer solution pH 7.4 and stirred at a rate of 150 rpm at room temperature , 15. MucPhysical characterizationAccording to the literature, the success of film formation is proved by the fact that the prepared films are smooth in texture, translucent and uniform without any visible cracks or imperfections , 17. RegUniformity of weightThe weight of ocular films in each batch was found to be uniform and in the range . The weiUniformity of thicknessThe thickness of ocular films in each batch varied in the range as expected . The forDrug content determinationThe drug content of ocular films has been presented in Swelling indexAs can be seen in Mucoadhesion studyThe mucoadhesion time of ocular films (in triplicate) on a freshly cut sheep eyelid was studied and it was proved that in all formulations with glycerin as plasticizer, mucoadhesion time was 10 hours. However, formulations with PEG 400 had no mucoadhesion. Meanwhile, utilizing TEC single resulted in a 3-5 hour mucoadhesion and when it was combined with glycerin (1:1), the time reached to 7 hours.In-vitro drug release studiesIn polymeric matrices, drug release is elicited by water accessibility into the matrix, breaking the polymer–polymer bonds and simultaneously leading to the formation of water–polymer bonds, which separates polymer chains, and swells to form a gel. The drug diffuses from gel network to the medium with a diffusion rate which is dependent on its diffusion ability through the gel and its concentration gradient. Concurrently, the rate of gel matrix erosion depends on medium hydrodynamics and molecular weight of polymer. Therefore, the drug release profile mainly depends on relative rates of these processes , and 17.in-vitro drug release studies (A1-D3). In In this study, all of the successfull ocular film formulations, with proper texture and thickness were subjected to 50% has been achieved in 4 hours and in ratio 1:1, t50% has increased to 5 hours and in ratio 3:1, this time has extended to 9 hours. Finally, the total amount of released drug was not more than 34% and even did not reach 50%. This can be explained by the hydrophobic nature of Eudragit comparing HPMC and is proved by swelling studies, entrapment of drug molecules in polymeric network and less accesibilty to water channels which lead to slower release rate. In Group D formulations . It was confirmed by zero order plots that the drug diffused slowly from ocular fims. In planar geometry by applying Korsmeyer-Peppas model, the value of n = 0.5 implies a Fickian diffusion, 0.5 < n < 1.0 indicates anomalous (non-Fickian) transport, and n = 1 indicates case II (relaxation controlled) transport. In the present films, this value was found in the range of 0.8473–1.0092 which implied that the release mechanisms followed anomalous (non-Fickian) transport and zero order release (case II transport)  .Various batches of diclofenac sodium ocular films applying hydrophylic HPMC and Eudragits were prepared using solvent casting method and then were evaluated.in-vitro over the period of 11 hours with zero order kinetics. D2b and D2c formulations containing Eudragit RS PO and HPMC with ratio 1:1 and glycerin as plasticizer satisfied all pharmaceutical parameters of ocular films and demonstrated the controlled release of the drug The results of the present study revealed that polymer types as well as their properties, drug: polymer ratio and plasticizer type to formulate the ocular films are important criteria which influence the film swelling and thus affect the drug release. Our study suggested that the mucoadhesive feature and the sustaining effect on drug release obtained by proper optimization to prepare suitable film can be exploited as a potential candidate to formulate sustained release diclofenac ocular films.


A minipig model was implemented to assess the bone healing through histology and mRNA expression at different time points . The novel ATZ implant was compared to a titanium clinical standard. The implants were analyzed in terms of microstructure and surface roughness before in vivo tests. The most interesting result deals with a statistically significant higher digital histology index for ATZ implants with respect to titanium standard at 56 days, which is an unprecedented finding, to the authors' knowledge. Even if further investigations are needed before proposing the clinical use in humans, the tested material proved to be a promising candidate among the possible ceramic dental implants.Ceramic materials are widely used for biomedical applications because of their remarkable biological and mechanical properties. Composites made of alumina and zirconia are particularly interesting owing to their higher toughness with respect to the monolithic materials. On this basis, the present study is focused on the  Titanium implants have the longest traceable record of predictable clinical performance with very high success rate ; however in vivo [Alumina and yttria stabilized zirconia (Y-TZP) ceramics are suitable for biomedical applications, due to their good mechanical and tribological properties and proved biocompatibility –14. Pure in vivo –22. Zirc in vivo  and the  in vivo .2 is kept under the 22% wt [To overcome the limits of both monolithic materials, researchers have focused on the preparation of composites made of alumina and zirconia –27, alree 22% wt , ageing The mechanical stability is not the only requirement a good implant material should possess, since bioactivity is necessary to ensure proper osseointegration. When dealing with bone bonding materials, bioactivity can be described as the ability to grow bonelike apatite on the material surfaces . Apatite in vivo behaviour of such material. For this purpose, implants made of ATZ were placed within the bone of recipient animals, using a titanium clinical standard for comparison since no papers on this topic are present in literature. For sake of completeness, fatigue tests were performed according to UNI EN ISO 14801:2008 standards and surface properties were studied. Indeed, as load bearing medical devices, dental implants are not suitable to clinical use if they present defects possibly affecting their mechanical strength, which may lead to early failure under the chewing load; such might be the case of large porosities within ceramic bulk materials.The aforementioned promising properties of ATZ treated with phosphoric acids, in terms of mechanical and bioactivity features, prompted the authors to study the2-20wt %Al2O3, (TZ-3Y20AB), as “ready to press” powders, so that no additional mixing was required before pressing. Green samples were obtained by linear pressuring at 80 MPa followed by Cold Isostatic Pressing under 200 MPa. The optimized conditions for sintering process were: heating 50°C/h up to 700°C, dwelling for 2 h at 700°C, and heating of 100°C/h up to temperature sintering of 1500°C and dwelling for 2 h at this temperature. Hardness, toughness, and strength of the full dense material was measured on proper specimens, as reported elsewhere by Faga et al. [Powders with high purity were used to produce the oxidic implants  of the three samples, was applied to the samples for 5 × 106 cycles in a sinusoidal way. The minimal strength Fmin corresponded to the 10% of the maximal strength Fmax .Mechanical tests were run in triplicate according to UNI EN ISO 14801:2008 by using a monoaxial machine for both the dynamic and static tests  equipped with a loading cell (max. load: 3 kN). In static conditions, the speed was 0.2 mm/minute, the preload: 2N. Based on the ISO standards mentioned above, the static tests aim to define the load to be applied when performing the dynamic tests. Therefore, a value inferior to the 80% of  in vivo experiment was conducted on 16 minipigs. Eight experimental implants per animal were inserted in the right tibia: 4 hydrothermally treated ATZ and 4 Nobel Ti-Unite . Four animals were sacrificed at 8, 14, 28, and 56 days after the implant placement. The tibias were block-sectioned and subjected to histomorphometric  and biomolecular analysis . Outcomes were analyzed in terms of new bone apposition by a digital histology index (DHI) and RNA profiling.The ad libitum. The animals underwent an acclimation period of 1 week prior to surgery. Abiding with Italian law, all animal experiments were approved by an academic ethics committee.Sixteen adult minipigs (mean weight 65.94 kg SD = 2.84) were used in the experiment . The minipigs were fed standard pelleted cereal food and were given water2. The right hind leg was prepared in a standard sterile fashion. After exposing the tibia, the implants were inserted with a 40 Ncm torque. Then, the flap was closed and the surgical access sutured so as to completely cover the implants, whose head reached the bone level. Each tibia received 8 implants: 4 ceramic ones and 4 titanium implants. Tibial bone specimens were collected at this stage to determine baseline (time 0) values for the RNA analysis. At the established time points, animals were euthanized by preanesthesia with 2% xylazine  (2.2 mg/kg) and tiletamine/zolazepam (6.6 mg/kg) and an intracardiac injection of embutramide, mebezonium iodide, and tetracaine hydrochloride (70 mg/kg). Finally, the tibias were exposed and dissectedinto slices.After preanesthetic sedation with 2% xylazine  and tiletamine/zolazepam , surgery was performed under intubation anesthesia with isoflurane/halothane and O Sus scrofa  assay ID: Ss03392006_m1; bone gamma-carboxyglutamate (gla) protein assay ID: Ss03373655_s1; bone morphogenetic protein 2 assay ID: Ss03373798_g1 Applied Biosystems, USA), following the manufacturer's protocol. Glyceraldehyde 3-dehydrogenase  was used as internal control.To protect the RNA, all specimens  were placed in RNA Later  and stored at −80°C until testing. Before the RNA purification , the samples were disrupted using a TissueRuptor. Total RNA was subjected to reverse transcription . The cDNA obtained underwent real-time polymerase chain reaction (RT-PCR) using commercially available primer/probe cocktails forμm thick sections, along the longitudinal implant axis, using a motorized microtome. Polylysine coated slides were used to enhance the adhesion of the tissue section during staining procedures. The histological structure of the peri-implant bone was assessed by traditional haematoxylin and eosin staining and for optical microscopy. The digital histology index (DHI) was manually determined using an imaging computer software (Olympus Dot Slide BX51) on the virtual histology slide. Briefly, on each virtual slide, the newly formed bone was measured by tracing a line at the interface between bone and implant within a given length (a standard length of 4 cm was adopted). The ratio of newly formed bone to the total bone-implant interface taken into consideration was expressed as a percentage. The DHI was measured on both sides of each slide. Two specimens per time points per material were obtained and at least 10 slides were made from each block sectioned histological sample. In addition, morphometric parameters such as (1) presence of necrotic or fibrous tissue and (2) amount of organized grouped osteoclasts and osteoblasts, together with (3) blood vessels and (4) de novo formed bone, were assessed by two independent histologists.To evaluate the bone healing and remodeling, histologic analysis was performed at 8, 14, 28, and 56 days after implant. After block section of the implants along with the adjacent bone, the specimens were fixed in 4% formalin for 24 hours and decalcified for 3 to 4 weeks in a mixture of 50% formic acid and 10% sodium citrate tribasic. While the implants were removed, the peri-implant bone samples were embedded with paraffin wax and cut into 3 μm thick, were cut from the 56-day-paraffin-embedded blocks and stained as described above. Afterwards, the slides were acquired by an automated microscope to generate virtual colored slides, which were converted into grey scale images so as to allow further image processing. The three-dimensional reconstruction of the samples was performed using Amira 4.0, an advanced volume modeling software . Three different volumes were delimitated from the cortex to the medullar space and a percentage of bone density was calculated based on a densitometric analysis of the white voxels, the cortical bone being taken as a reference (100%).Forty serial sections, 3 n = 3), on the samples derived from peri-implant bone . As for the DHI, each specimen  generated at least 10 virtual histological slides. Statistical analysis was performed by using the one-way analysis of variance (ANOVA) with post hoc Dunnett's test or the Student's t-test, as appropriate. A P value of <0.001 was considered significant.Data from RT-PCR and DHI were analysed by GraphPad Prism 5 . RT-PCR was independently repeated at least three times  for the bone markers observed  in peri-implant bone tissue .Presence of osteoid at the bone-implant interface was noted at the earliest (8 days) time point whereas little or no interfacial unmineralized matrix was seen at all other time points for both ATZ and titanium hosting bones. A minimal amount of necrotic osteocytes were noted only at 8 days in proximity to the implant surface in both groups , at the Both treatment and control groups showed a steady increase in the overall digital histology index up to 56 days . The DHIt-test P < 0.001).The densitometric analysis of the white voxels of the medial and the apical areas of the samples concerning the 56th day is reported in  in vivo experiment portrayed in the present paper on the basis of previous data dealing with the bioactivity assessed as per Faga et al. [There is still a huge concern about the long-term durability of the Y-TZP , due to the low temperature degradation (LTD) of zirconia –19, despa et al. . Indeed,a et al. .μm) higher than that of the titanium implants (Sa = 3.4 μm). Topography and surface roughness are known to positively affect the healing process [At the end of the manufacturing process, the ATZ implants were analysed by scanning electron microscopy and submitted to profilometry for roughness evaluation, before the placement into the recipient animals, adopting a swine model previously described . As a co process –46. Inde process , as it w process .Biomolecular, histological and histomorphometrical analyses were used to examine the differences in the healing and remodeling processes between the two different implant materials. Four different time points were evaluated. Representative of the early phase of healing were 8 and 14 days, while intermediate and mature bone healing were reasonably foreseen at 28 and 56 days, respectively, based on earlier experiments . Interes in vitro, even in absence of modifications and when roughness is excluded by mirror polishing [Although ATZ is able to elicit a satisfying biological responseolishing , no signBased on this study, further investigations are needed before recommending the clinical use in humans, although the tested material proved to be a promising candidate among the possible ceramic dental implants.


For patients who experience dry eye after phacoemulsification, vision and quality of life can significantly deteriorate. In this study, the efficacy and safety of carboxymethylcellulose sodium (CMC) 1% ophthalmic solution combined with conventional therapy in treating dry eye signs and symptoms after phacoemulsification were evaluated.In this prospective, multicenter, open-label, controlled study, 180 patients with age-related cataract were randomized to treatment with conventional therapy plus CMC 1% (n = 90) or to conventional therapy only  after phacoemulsification and intraocular lens implantation. Tear breakup time (TBUT), the Schirmer test with anesthesia, and fluorescein and lissamine green staining were performed. The Ocular Surface Disease Index (OSDI) questionnaire and a patient subjective symptom evaluation were administered preoperatively (baseline) and postoperatively at 7 and 30 days.P = 0.0475) and day 30  after surgery. Compared with baseline, TBUT significantly increased in patients in the treatment group (P < 0.001 at both day 7 and day 30) with a presurgical diagnosis of dry eye, but significantly decreased in patients in the control group (P < 0.02 at both day 7 and day 30) with no prior diagnosis of dry eye. Fluorescein and lissamine staining, OSDI questionnaire and subjective symptom scores all improved from baseline, with no significant differences between the two groups. No significant differences in tolerability and safety were observed between the group receiving CMC and conventional therapy, and those receiving conventional therapy only.TBUT was significantly longer in the treatment group compared with the control group at day 7 .ClinicalTrials.gov identifier  Dry eye is a multifactorial disease of the tears and ocular surface that results in symptoms of discomfort, visual disturbance, and tear film instability, with potential damage to the ocular surface ,2. HighlWith the advancement of medical technology, cataract surgery (phacoemulsification with intraocular lens [IOL] implantation) has become a routine procedure; however, for patients who experience dry eye after phacoemulsification, vision and quality of life can significantly deteriorate. Clinical evaluation of dry eye after cataract surgery has demonstrated worsening of patient subjective symptom scores, reductions in tear film breakup time (TBUT) and goblet cell density, as well as increased corneal and conjunctival staining, indicating deteriorating disease -7. VarioConventional therapy following cataract surgery includes topical corticosteroids or non-steroidal anti-inflammatory drugs to manage inflammation, and anti-infectives. The effectiveness of corticosteroids in managing dry eye after cataract surgery has been evaluated ,10. HoweThis prospective, open-label, interventional, randomized, controlled study was conducted at eight clinical sites in China between October 2011 and May 2013. The study protocol was approved by the central ethics committee  and two branch ethics committees . The study was conducted in accordance with the principles of the Declaration of Helsinki, and all patients signed an informed consent form before enrollment.Male or female patients aged 60 to 80 years with age-related cataract were enrolled. Patients were scheduled to undergo phacoemulsification and IOL implantation and had a lens nucleus hardness of grade 3 or less (based on nucleus color per the standard Lens Opacities Classification System III ). ExcludPatients who provided informed consent were enrolled in the study by their treating physician, and were assigned a sequential number with a corresponding randomization code generated by an independent third party using SAS software . According to the randomization code, clinical staff assigned patients to receive either study treatment plus conventional post-surgical therapy consisting of (1) prednisolone acetate ophthalmic suspension 1%  instilled four times daily in the first post-surgical week, three times daily in the second week, twice daily in the third week, and once daily for the remainder of the study, and (2) levofloxacin 0.5% ophthalmic solution , or conventional therapy alone (control group) instilled four times daily in the first post-surgical week. The clinical staff provided treatment medications and instructions on how to administer ophthalmic solutions according to the assigned randomization group.Topical anesthesia with oxybuprocaine hydrochloride 0.4% eye drops  or proparacaine hydrochloride 0.5% eye drops  was administered before the surgical procedure. After surgery, the study treatment group instilled CMC sodium 1% ophthalmic solution  four times daily, along with conventional post-surgical therapy for the first 30 days. The control group received conventional post-surgical therapy only. During the study, use of other topical ophthalmologic agents with a potential impact on efficacy assessment of the study treatment was prohibited.®, Alcon Laboratories, Inc) as anesthetics . The Ocular Surface Disease Index (OSDI) questionnaire and patient subjective symptom evaluation were also completed at each visit, prior to the above tests.Patients were evaluated 3 to 5 days before phacoemulsification and IOL implant surgery (baseline), as well as 7 and 30 days post-surgery to assess the benefit of CMC 1% at early and later time points. On the day of each of the three visits, administration of CMC was withheld prior to and immediately after study assessments. The following were conducted in sequence: TBUT, fluorescein and lissamine green ocular staining, and Schirmer test with oxybuprocaine hydrochloride 0.4% eye drops  or proparacaine hydrochloride 0.5% eye drops  on the lower eye lid. Five regions, the temporal and nasal conjunctival regions and three corneal regions , were assessed. Corneal and conjunctival were graded in each region as 0 = no staining; 1 = staining less than half the area; 2 = staining an area greater than half but not the whole region; and 3 = staining of the whole region. The Schirmer test was performed to evaluate basic secretion from the lacrimal glands .The standard OSDI questionnaire and a patient subjective symptom evaluation form were used to evaluate dry eye symptoms. In the OSDI questionnaire, 12 questions assessed visual function, ocular symptoms, and potential environmental triggers. The subjective evaluation form included 11 symptoms: foreign body sensation, photophobia, itching, pain in the eyes, dry eyes, eye heaviness, blurred vision, eye fatigue, eye discomfort, eye secretions, and tearing. Each symptom was graded as 0 = no symptom; 1 = occasional symptoms; 2 = intermittent mild symptoms; and 3 = persistent clear symptoms.The clinical investigator was responsible for monitoring adverse events  and reporting incidents to the study sponsor. Any worsening of pre-existing dry eye symptoms  or new occurrence of such symptoms was also considered an adverse event.Patients were diagnosed as having dry eye symptoms if they met one of the following conditions: TBUT ≤5 s, plus a subjective symptom score of 3 or greater ,15, or St-test  and the Wilcoxon rank sum test . Similarly, differences before and after surgery within the treatment and control groups were assessed using paired t-tests  and Wilcoxon signed rank test . P < 0.05 was considered to be statistically significant. Statistical analysis was performed using SAS (version 8.0).Quantitative data were compared between the two groups using the Sample size for the study was based on results from an early study, in which the value of TBUT was determined to be 4.2 ± 2.7 s in the control group and 7.3 ± 4.9 s in the artificial tear group at day 7 after cataract surgery . One-taiA total of 180 patients were enrolled and formed the ITT population—90 patients were randomized to each study group. Among the enrolled patients, seven patients in the treatment group and eight patients in the control group did not complete the study  .Based on the criteria for diagnosis of dry eye in this study, the percentage of patients considered to have dry eye disease before surgery was similar in both the treatment and control groups. At day 7 after surgery, the number of patients with dry eye decreased to 22 (24.4%) in the treatment group but increased slightly to 35 (38.9%) in the control group (P = 0.0114). At day 30, no significant differences were found in the number of patients with post-surgical dry eye in the two groups (17 [48.6%] versus 20 [60.6%]). In the subgroup of patients without dry eye before surgery, no differences were observed between the two groups at both day 7 (6 [10.9%] versus 10 [17.5%]) and day 30 (8 [14.5%] versus 11 [19.3%]) post-surgery.Among the group of patients with dry eye before surgery, a significantly lower number of patients continued to have dry eye after surgery in the treatment group  compared with the control group  at day 7  was significantly greater in the treatment group than the control group at both day 7 and day 30 Figure . CompareP < 0.001). In contrast, no improvements in TBUT were observed in the control group up to 30 days after surgery  between the two groups at both day 7 and day 30 after cataract surgery (Table P < 0.001); no significant changes were observed in the control group.No differences in fluorescein staining scores (mean ± SD) were observed between the treatment and control groups at either day 7 or day 30 post-surgery. However, fluorescein scores significantly decreased from baseline at day 30 in the treatment and control groups (Table P = 0.0233) and at day 30 in the treatment group .Lissamine green staining scores (mean ± SD) were not significantly different between the two groups at day 7 and day 30 post-surgery. Similar to fluorescein scores, there were significant decreases from baseline in lissamine green scores at day 30 in both the treatment and control groups (Table P = 0.0466).No differences were reported in OSDI and patient subjective symptom scores (mean ± SD) between the treatment group and control group at day 7 and day 30 following cataract surgery. However, both OSDI and subjective symptom scores significantly decreased from baseline at day 7 and day 30 post-cataract surgery in both groups Table . In subgA total of 171 patients, 86 in the treatment group and 85 in the control group, formed the safety analysis set. No significant differences were noted in safety between the groups; 5 (5.8%) patients in the treatment group and 4 (4.7%) patients in the control group experienced adverse events  in the control group. The findings indicate that cataract surgery may worsen dry eye (as has been previously reported), and further suggest that the addition of CMC 1% to conventional post-surgical therapy may help prevent development/worsening of dry eye after phacoemulsification. Since neither patients nor investigators were masked to treatment, patients in the control group were likely aware of their treatment status, which could explain the above findings. However, despite this potential limitation of the study, the difference in the percentage of patients with dry eye between the treatment and control groups was not significant by day 30 after surgery, arguing against this possibility. One possible, alternative explanation for this observation is that the ocular surface may have recovered from damage caused by phacoemulsification. Consistent with this possibility, the post-surgical decrease in symptoms observed in the treatment and control groups may be attributable to enhanced vision as a result of phacoemulsification, although it is also possible that the questionnaires used were not optimally designed to assess dry eye following cataract surgery.CMC is a polymer composed of glucopyranose subunits with anionic charge and high microviscosity properties, which allow retention on the cornea for a prolonged time period. An in vitro study reported that CMC may remain bound to human corneal epithelial cells (HCECs) for 2 h, and that CMC may bind to HCECs through interactions between glucopyranose subunits and glucose transporters . A singlIt is unclear why post-surgical TBUT values observed in this study were similar in the treatment and control groups of patients who had dry eye prior to surgery. However, since steroids can reduce inflammation in the injured ocular surface epithelium of patients with dry eye, and prednisolone was part of the post-surgical therapy administered to the entire study population, it is possible that prednisolone prevented the detection of a difference between the treatment and control groups. Nonetheless, at day 30 post-cataract surgery, TBUT decreased from baseline in the control group, but improved in the treatment group. This finding is consistent with that of a previous study, which demonstrated that CMC prolonged TBUT in patients with mild to moderate dry eye . Our stuAs increasing evidence supports the hypothesis that ocular surface and lacrimal gland inflammation plays a central role in the pathogenesis of dry eye ,26, the Although this study was limited in that neither patients nor investigators were masked to treatment, results indicate that adding CMC 1% ophthalmic solution to conventional therapy after phacoemulsification can help manage patients with prior dry eye disease. This combination can also maintain tear film stability and TBUT in patients without dry eye disease prior to phacoemulsification. Further studies are required, designed to determine mechanisms in which CMC helps stabilize the tear film after cataract surgery.