Based on their ability to recognize and eliminate various endo- and exogenous pathogens as well as pathological alterations, Natural Killer (NK) cells represent an important part of the cellular innate immune system. Although the knowledge about their function is growing, little is known about their development and regulation on the molecular level. Research of the past decade suggests that modifications of the chromatin, which do not affect the base sequence of the DNA, also known as epigenetic alterations, are strongly involved in these processes. Here, the impact of epigenetic modifications on the development as well as the expression of important activating and inhibiting NK-cell receptors and their effector function is reviewed. Furthermore, external stimuli such as physical activity and their influence on the epigenetic level are discussed. Natural killer cells (NK cells) are historically named by their ability to kill target cells without prior priming on a “natural” way . As a pa− CD56+) [bright and a CD56dim subgroup. CD56bright NK cells express high amount of the NK cell marker CD56 and are characterized by a lower cytotoxic capacity but high secretion of cytokines upon stimulation [bright NK cells represent the minority of NK cells and are mainly located in secondary lymphoid tissues [dim NK cells express CD56 in low amounts on the cell surface and display a high cytotoxic capacity [dim are the majority of NK cells circulating through the body [Their phenotype is determined by surface expression of the NK cell marker CD56 and a concomitant absence of the T-cell marker CD3 (CD3− CD56+) . In genemulation ,2. CD56b tissues ,7. In cocapacity ,2. With the body ,7.+ NKG2Chi NK cells has adaptive functions and mediates a fast recurring response against viral infections like Cytomegalo Virus (CMV) [Current literature suggests a third subgroup of NK cells, described as memory NK cells. This population of CD57us (CMV) .The effector function of NK cells is regulated by an orchestra of activating and inhibitory receptors on the cell surface. These receptors are encoded by the germ line and recognize structures of high molecular weight . All inhThe activation of NK cells effector function is mediated by a critical balance of signals from inhibitory and activating receptors. When an activation of effector function is achieved, NK cells secrete granules containing effector molecules like perforin and granzyme B. Perforin is a 70 kDa protein containing a conserved membrane attack complex of Complement/Perforin (MACPF) domain . The MACIn 1942, about 20 years before the Nobel-Prize for defining the ultrastructure of the DNA was awarded, Conrad Waddington defined epigenetic as “The branch of biology which studies the causal interactions between genes and their products, which bring the phenotype into being”. Newer definitions of epigenetic describe it as heredity which is independent of the DNA sequence and as research of changes in gene expression and mitotic heredity of gene expression patterns . The actDNA methylation is catalyzed by DNA-methyl-transferases (DNMTs) by the addition of a methyl-group on cytosines in the 5th position. Therefore, DNA methylation occurs at cytosine-guanine-dinucleotides (CpGs) and mostly in regions with a high CpG density. At the beginning of epigenetic, research DNA methylation was described to be inhibitory for gene expression. It is described that methylation of CpG rich regions could inhibit the binding of transcription factors that recognize these regions  and furtHistone modifications result in opening or closing the chromatin structure. Depending on the posttranslational modification, repression or activation of gene transcription is achieved. For example, the acetylation of lysine residues on histone 3 and 4 is associated with active transcription, whether methylation of lysine residues on these histones could be activating or inhibitory . The mosFinally, the expression of microRNA molecules is described as epigenetic mechanism. These small endogenous, single-stranded RNA molecules modulate gene expression by binding to complementary sites in the 3′-untranslated region (3′-UTR) of the target genes mRNA . This inet al. [− Lymphocytes. In contrast, NK cells and other KIR expressing Lymphocytes reveal a demethylation of KIR genes that resulted in the abolishment of KIR gene expression. Similar results were reported by Gao et al. [et al. [Only a few studies have investigated the influence of epigenetic modifications on the differentiation and maturation of NK cells. Santourlidis et al.  showed to et al. , who des [et al.  found th [et al. . Further [et al. . MiR-181 [et al. . Notch s [et al. ,24.+ and NKG2Chi [Regarding epigenetic modifications during differentiation and maturation, most studies focused on memory NK cells which display elements of the adaptive immune system and are induced by an infection with cytomegalovirus (CMV). Memory NK cells have been discovered in mouse models and have been described to have properties of memory T cells, such as being self-renewing, long-lived and to show expansion upon a second viral challenge . These c NKG2Chi  as well  NKG2Chi . Deficie NKG2Chi , which i NKG2Chi  showed s+ and CD4+ T cells, what resulted in higher similarities between memory NK cells and CD8+ and CD4+ T cells than between memory and canonical NK cells [H1 cells [Furthermore, the DNA methylation profile of canonical and memory NK cells was compared to CD8NK cells . The comNK cells . ImprintAs mentioned above, NK cell activity is related to a complex interaction of activating and inhibiting receptors on the NK cell surface. Interestingly, both activation patterns (cytokines or secretion of cytotoxic agents) seem to be epigenetically regulated.et al. [Incubation with HDAC inhibitors (HDACi) like suberoylanilide hydroxamic acid (SAHA) or valproic acid (VPA) suppresses Interleukin (IL)-2-activated NK cell cytotoxicity . These ret al.  who founet al. [Besides histone modifications, the DNA methylation is important to orchestra the NK cell effector function. To investigate the effect of DNA methylation, demethylating agents like 5-azacytidine (Aza) and decitabine (Deci) are used. Aza is an analogue to the DNA base cytidine, whereas Deci is the analogue to desoxycytosine. Schmiedel et al.  describeet al. ,29 and aet al. ,29. The et al. .et al. [et al. [et al. [It has to be noted, that IL-2 seems to be a cofactor for DNA demethylating or histone acetylating agents. While Gao et al.  and Schm [et al.  used IL- [et al.  tried th [et al. , wherebyAs mentioned above, epigenetic modifications are involved in NK cell differentiation and regulation of effector function. NK cell receptors are differentially expressed through the development of HPCs to mature NK cells and are crucially involved in the induction and inhibition of NK cell effector function.et al. [The suppression of NK cell cytotoxicity by incubation with HDACi observed by Ogbomo et al.  was due KIR belong to the Ig superfamily and are named by the number of extracellular immunoglobulin domains  and their type of signaling, which can be stimulatory (S) or inhibitory (L) .et al. [+ and KIR3DL1− NK cells showed a densely methylated KIR3DL1 promoter in KIR3DL1− cells and a completely unmethylated promoter in KIR3DL1+ cells [et al. [Gao et al.  investiget al. ,31. Furt1+ cells ,32. Theset al. [During the development of HPCs to mature NK cells, the DNA demethylation of KIR genes leads to KIR expression. But DNA methylation does not just determine which KIR gene is expressed, it also determines which allele expresses the KIR gene. KIR genes are mostly expressed in a mono-allelic manner with non-expressed alleles exhibit DNA hypermethylation and expressed alleles DNA hypomethylation . Furtheret al.  mentioneet al. [Besides chromatin modifications, KIR genes are also regulated by microRNA. As reported by Davies et al. , KIR genet al. .et al. [Cichockie et al.  describeet al. .In contrast to DNA methylation, histone acetylation seems to play a less important role in KIR gene regulation. Incubation of NK cells with HDACi did not affect KIR gene expression ,31. Moreet al. [In addition to KIR gene regulation, epigenetic modifications have been reported to be involved in the expression of NKG2D, which is one the most important activating NK cell receptor. Fernandes-Sanchez et al.  found a et al. . Furtheret al. . Furtheret al. , is unknet al. [et al. [Besides histone modifications and DNA methylation, microRNAs are involved in the regulation of NKG2D expression. Espinoza et al.  have shoet al. . Intereset al. . This im [et al.  describeet al. [Life style factors such as physical (in-) activity and nutrition are supposed to influence the immune system and its function . NK cellet al. ).et al. [Since physical activity and exercise are known to induce short- and long-term epigenetic alterations in various cell types and tissues ,38,39,40et al.  who haveet al. focused on changes in histone acetylation and DNA methylation in NK cells in response to exercise. In a first study, the influence of chemotherapeutic treatment and a single bout of exercise on global H3K9Ac and H4K5Ac in NK cells of B-cell non-hodgkin lymphoma patients and a healthy control group has been determined [et al. [Against this background, Zimmer termined . After ttermined , which ltermined . Interes [et al.  who demoAnother mechanism by which regular physical activity may alter the epigenome of NK cells is a reduction of psychophysiological stress which is associated with the secretion of glucocorticoids . As reviet al. [et al. [et al. [Krukowski et al.  revealed [et al.  confirme [et al. . Therefo [et al.  and conc [et al. , it mighAll studies mentioned above also analyzed the histone acetylation at the promoter regions of perforin and granzym B and could show that treatment with Dexa resulted in a reduced histone acetylation ,47,48 whex vivo experiments. Therefore, functional alterations of NK-cells which were detected in such experiments are hard to transfer from bench to practice. Translational research approaches combining basic research findings with clinical outcomes  are necessary to approve results from ex vivo experiments. Further research is needed to fulfill the understanding of the epigenetic regulation of NK cells and potential therapeutic implications.Epigenetic modifications seem to play a key role in NK cell development and regulation. On the one hand, a stable epigenetic imprinting is used for maintaining the way of differentiation. On the other hand, the dynamic properties of epigenetic modifications are used for adapting/reacting on internal and external stimuli . However, the significance of the described findings is limited by study designs. Although human NK-cells were frequently used, in most cases treatment took place in


The study develops an integrated humidity microsensor fabricated using the commercial 0.18 μm complementary metal oxide semiconductor (CMOS) process. The integrated humidity sensor consists of a humidity sensor and a ring oscillator circuit on-a-chip. The humidity sensor is composed of a sensitive film and branch interdigitated electrodes. The sensitive film is zinc oxide prepared by sol-gel method. After completion of the CMOS process, the sensor requires a post-process to remove the sacrificial oxide layer and to coat the zinc oxide film on the interdigitated electrodes. The capacitance of the sensor changes when the sensitive film adsorbs water vapor. The circuit is used to convert the capacitance of the humidity sensor into the oscillation frequency output. Experimental results show that the output frequency of the sensor changes from 84.3 to 73.4 MHz at 30 °C as the humidity increases 40 to 90 %RH. Liang et al. [2O3 film and Pt interdigitated electrodes. The sensing material of ZnO-In2O3 was deposited by radio-frequency sputtering.Humidity sensors are widely used in industrial, electronic, and biomedical equipment. Conventional humidity sensors have the disadvantages of large volume and high cost. On the contrary, the advantages of humidity microsensors include small volume, low cost, high performance and easy mass-production . Recentlet al.  proposed [et al.  also useg et al.  presenteet al. [et al. [et al. [et al. [et al. [et al. [et al. [Zinc oxide can be applied as a piezoelectric, gas-sensing and photoelectric material. Many studies have utilized zinc oxide as the sensitive material of humidity microsensors. For instance, Zhang et al.  fabricat [et al.  presente [et al.  employed [et al.  used a h [et al.  proposed [et al. –9 are su [et al. –9 were n [et al. . In this [et al. ,6,8,9, aet al. [The commercial CMOS process has been utilized to manufacture various microactuators and microsensors ,11. Micret al. , was fabet al. –9. The cet al.  to coat 2.2. The sensitive film of the sensor is zinc oxide, and the film is coated on the interdigitated electrodes. When the sensitive film absorbs or desorbs humidity vapor, the sensor produces a variation in capacitance.sensorf of the ring oscillator circuit is given by sensorτ the delay time associated with the humidity sensor; invτ is the delay time associated with the inverters; sensorC is the humidity sensor capacitance; loadC is the load capacitance; and ΔV and aveI are the threshold voltage and average current, respectively. According to 3.iso-propanol (100 mL), and the mixture was denoted solution A. Sodium hydroxide (0.5 g) and poly(vinyl pyrrolidone) (2 g) were added to iso-propanol (50 mL), and the misture was denoted solution B. Solution B and hexamethylenetetramine (0.7 g) were added to solution A with vigorous stirring at 75 °C for 2 h. The mixture was transferred into a teflon-lined stainless steel autoclave, sealed and maintained at 120 °C for 12 h. After the reaction, the resulting products were filtered, and washed with deionized water and ethanol. Finally, the zinc oxide film was coated on the substrate, followed by calcination at 350 °C for 2 h.The sensitive material of zinc oxide was prepared by the sol-gel method. The preparation steps were as follows : zinc acThe surface morphology of the zinc oxide film was measured by scanning electron microscopy . 4.The integrated humidity sensor chip was manufacture using the commercial 0.18 μm CMOS process of TSMC . 5.A spectrum analyzer, a test chamber  and an LCR meter were employed to test the characteristics of the integrated humidity sensor. The capacitance variation of the humidity sensor was measured by the LCR meter. The output frequency of the humidity sensor was recorded by the spectrum analyzer. The humidity and temperature of the test chamber could be tuned. The test chamber could supply a humidity range of 30–95 %RH and a temperature range of 25–100 °C.To understand the capacitance variation of the humidity sensor, the sensor without the ring oscillator circuit was tested under different humidity. The humidity sensor without the circuit was set in the test chamber. The test chamber provided different humidity to the sensor, and the LCR meter recorded the capacitance variation of the sensor. The output frequency of the humidity sensor with the ring oscillator circuit was measured. The ring oscillator circuit converted the capacitance variation of the humidity sensor into the oscillation frequency output. The sensor with the circuit was set in the test chamber. The power supply provided a bias voltage of 3 V to the circuit. The spectrum analyzer detected the output frequency of the sensor. To characterize the influence of temperature, the integrated humidity sensor was tested under different temperatures. et al. [et al. [et al. [Hu et al.  reportedet al. . Dai [21 [et al.  presente [et al. .6.An integrated humidity sensor has been fabricated using the commercial 0.18 μm CMOS process and a post-process. The integrated humidity sensor contained a humidity sensor and a ring oscillator circuit. The humidity sensor was a capacitive type. The sensor generated a change in capacitance when it sensed water vapor. The ring oscillator circuit converted the capacitance variation of the sensor into the output frequency. The humidity sensor consisted of branch interdigitated electrodes and a sensitive film. The sensitive film was zinc oxide that prepared by the sol-gel method. The post-process included a wet etching to remove the sacrificial oxide layer and a zinc oxide film to coat on the interdigitated electrodes. The experimental results revealed that the output frequency of the sensor changed from 84.3 to 73.4 MHz at 30 °C when the humidity increased 40 to 90 %RH.


Cell adhesion is essential in cell communication and regulation, and is of fundamental importance in the development and maintenance of tissues. The mechanical interactions between a cell and its extracellular matrix (ECM) can influence and control cell behavior and function. The essential function of cell adhesion has created tremendous interests in developing methods for measuring and studying cell adhesion properties. The study of cell adhesion could be categorized into cell adhesion attachment and detachment events. The study of cell adhesion has been widely explored via both events for many important purposes in cellular biology, biomedical, and engineering fields. Cell adhesion attachment and detachment events could be further grouped into the cell population and single cell approach. Various techniques to measure cell adhesion have been applied to many fields of study in order to gain understanding of cell signaling pathways, biomaterial studies for implantable sensors, artificial bone and tooth replacement, the development of tissue-on-a-chip and organ-on-a-chip in tissue engineering, the effects of biochemical treatments and environmental stimuli to the cell adhesion, the potential of drug treatments, cancer metastasis study, and the determination of the adhesion properties of normal and cancerous cells. This review discussed the overview of the available methods to study cell adhesion through attachment and detachment events. Adhesion plays an integral role in cell communication and regulation, and is of fundamental importance in the development and maintenance of tissues. Cell adhesion is the ability of a single cell to stick to another cell or an extracellular matrix (ECM). It is important to understand how cells interact and coordinate their behavior in multicellular organisms. In vitro, most mammalian cells are anchorage-dependent and attach firmly to the substrate . AccordiSrc and Ras oncogenes reduces the adhesiveness to fibronectin (Fn) by impairing α5β1 integrins, the activation of oncogene ErbB2 in breast cancer up-regulates α5β1 integrin and enhances adhesion . Th. Thin viThe mechanical interactions between a cell and its ECM can influence and control cell behavior and function. The essential function of cell mechanobiology and its progressively important role in physiology and disease have created tremendous interests in developing methods for measuring the mechanical properties of cells. In general, cell adhesion studies can be categorized into cell attachment and detachment events. Numerous techniques have been developed to analyze cell adhesion events through the study of single cells as well as the populations of cells. Cell adhesion attachment events are focusing on the cell attachment mechanism to the substrate, while the detachment events involve the application of load to detach the adhered cells on the substrate .Cell attachment studies cover the analysis from the formation of a molecular bond between the cell’s surface receptors and the complementary ligands (on the ECM’s surface) to the observation of a population of cells’ responses through the cells’ behavior and changes of morphology during the attachment events. In cell migration, the cell adhesion plays a pivotal role in the driving force production . The adhPolyacrylamide-Traction Force Microscopy (PA-TFM). Numerous techniques have been developed to understand cell adhesion by characterizing single cells during their attachment events. PA-TFM is one of the widely used techniques to study single cells’ traction force, the force exerted by cells through contact to the substrate surface. Cells will be cultured on the polyacrylamide gel functionalized with the cells’ adhesive ligands and fluorescent beads embedded near the gel surface [et al. [et al. [et al. [MCF10A on PA substrates with a range of substrate compliances. Their findings showed that the wound could initiate a wave of motion that directs cells’ coordination towards the wound edge and substrate stiffness influenced the collective cell migration speed, persistence, and directionality as well as the coordination of cell movements [et al. [ surface . Upon th surface ,48,49. R [et al.  reported [et al.  improved [et al.  to studyovements . Tractio [et al.  reported [et al. .Micropatterning. Micropatterning  or micropillar) is a method that provides a micrometer scale: a soft, three-dimensional complex and dynamic microenvironment for both single cell studies and also for the multi-cellular arrangements in populations of cells. It relies on basic elastic beam theory, which makes force quantification easier and more reliable, as there is only one traction force field for each micropost/micropillar displacement map [et al. [et al. [N-isopropyla-crylamide) (PNIPAM) as an actuator which induces cell detachment when the temperature is reduced below 32 °C. It has been reported that the micropatterning technique is able to independently tune the biochemical, mechanical, and spatial/topography properties of biomaterials that could provide the opportunity to control cell fate for tissue engineering and regenerative medicine applications [ment map . Cell miment map . At the ment map . Micropa [et al.  found th [et al. . Mandal  [et al.  introducications . Laminarications .Three-Dimensional Traction Force Quantification (3D-TFM). The ability to grow cells within ECM gels (3D culture) is a major advantage to understand in vivo cellular cell behaviors, ranging from differentiated function to maintenance of stem cell functions [et al. [MDA-MB-231 breast carcinoma, A-125 lung carcinoma) and found that the directionality is important for cancer cell invasion rather than the magnitude of traction, and the invasive cells elongated with spindle-like morphology as opposed to the more spherical shape of non-invasive cells [βPix with srGAP1 that is critical for maintaining suppressive crosstalk between Cdc42 and RhoA during 3D collagen migration. Fraley et al. [unctions ,62. The unctions ,65,66,67unctions . Koch et [et al.  used theve cells . The disve cells  managed Wash Assay. In the population cell adhesion studies, the process of cell attachment can be divided into two types; static culture and dynamic culture, depending on the cell adherence mechanism during the cell culturing. Static culture is the stagnant condition of the cell culture medium during the incubation for cell adhesion, which is applicable for the culturing of cells inside microwell plates  is one of the widely used piezoelectric acoustic wave resonator [esonator  biosensoesonator . The senesonator  upon celesonator ,83,84,85esonator . The adhesonator ,83,86,87et al. [f) and acoustic wave energy dissipation can be used to gain direct measurements of the physical properties of the layers in contact with the chip [et al. [MC3T3-E1 and NIH3T3 cells on different precoated biocompatible surfaces. Braunhut et al. [et al. [et al. [Zhu et al.  found thet al.  reportedet al. ,88. The the chip ,88,89. T [et al. ,85 analyt et al. ,91,92,93t et al. . Recentl [et al.  have dev [et al.  producedMicrofluidics. In contrast to the static culture, the dynamic culture applies fluid movement during the cell culturing and adhesion process. Low fluid shear flow is needed to help the cell attachment process as it mimics the blood flow in the human body. Cells are continuously exposed to hemodynamic forces generated by blood flow in most biological systems. The balance between the adhesive forces generated by the interactions of membrane-bound receptors and their ligands with the dispersive hydrodynamic forces determines cell adhesion [et al. [et al. [et al. (2007) to analyze the deformation and biological and migratory capability of various tumor cell lines  to the lining of HMEC cells inside the channels [et al. [et al. [et al. [et al. [MDA-MB-231 cancer cells by analyzing the cells’ adhesion capability to the endothelial monolayer inside the channel.adhesion . Cell ad [et al.  reported [et al.  applied channels . Nalayan [et al.  have bui [et al.  have dev [et al.  studied  [et al.  to demonCell adhesion detachment studies involve load application to the adhered cells on the ECM to free the cells from their cell-matrix bonding . The appCytodetachment. Cytodetachment technique uses an atomic force microscopy (AFM) probe to physically detach individual cells in an open medium environment such as petri dish [et al. [L929 on four different materials by using cytodetachment technique. Using an image analysis system and fiber optic sensor, the apparent cell adhesive area was measured and the adhesive strength and detachment surface energy were calculated by dividing them by the cell adhesive area [NHIK 3025) were found to attached stonger and faster to the hydrophilic substrate using technique 1 at 37 °C when compared to 23 °C. There are multiple adhesion measurement studies combining the cytodetachment method with the laser tweezer work station [et al. [et al. [hFOB) cells was influenced by the cell’s shape grown on the Ca-P grooved micropattern surface.tri dish . Cells htri dish . Yamamot [et al.  studied ive area . Their five area . Human c station  and opti station ,105. The [et al.  used rab [et al.  reportedMicropipette Aspiration Technique. Micropipette aspiration is a widely used technique for measuring the mechanical properties of single cells. For single cell adhesion measurement, this technique detaches an immobilized cell by applying suction force to a portion of the cell surface employed by micropipette suction [et al. [−12 N [et al. [ suction  under ob suction . Adhesio suction , the cor suction , and the suction . Gao et  [et al.  used cel. [−12 N . Micropi [et al.  have dev [et al. .Single Cell Force Spectroscopy (SCFS). Force spectroscopy measurement methods were developed to measure the strength of cell adhesion down to single cell level. Commonly, the methods will use a microscope to observe the cell while force is applied to detach the cell using a nano/micromanipulator or micropipette. The imaging mode is used to study the structures and mechanics of isolated biomolecules [olecules ,111,112,olecules ,114, andolecules ,115, whiAFM Probe Force Measurement. AFM probe force measurement is widely used to measure the stiffness [et al. [Wnt11 signaling in modulating cell adhesion at single cell level. Weder et al. [Saos-2) during different phases of the cell cycle and found that the cells are loosely attached to the substrate during the cells’ round up (M phase) compared to during the interphase. Hoffmann et al. [NK cell receptor 2B4 on the early adhesion processes of NK cells using AFM-probe SCFS. In addition, AFM is flexible and can be integrated with the standard modern inverted and transmission optical microscope. Lee et al. [NIH/3T3 fibroblast cells and found the cell adhesion strength increased with culturing time, and growth factor was found to enhance the adhesion strength between the cell and substrate. The study was continued with the combination of the optical trapping technique to further study the single cell adhesion properties of MCDK cells in different phases of adhesion [MDCK cells through all the different phases of cell adhesion. Beaussart et al. [Staphylococcus epidermidis and Candida albicans.tiffness ,118,119 tiffness ,105,120 tiffness . This prtiffness . When thtiffness . Various [et al.  reportedr et al.  studied n et al.  determint et al.  studied Biomembrane Force Probe (BFP). BFP is a sensitive technique that allows the quantification of single molecular bonds. It is a versatile tool that can be used in a wide range of forces (0.1 pN to 1 nN) and loading rates (1–106 pN/s) [et al. [−2 to 103 picoNewton (pN) for probing molecular adhesions and structures of a living cell interface [et al. [06 pN/s) . This te06 pN/s) . The pro [et al. ,127 deventerface  to impronterface . Gourier [et al.  proved tOptical Tweezers (OT). OT uses a highly focused laser beam to trap and manipulate microscopic, neutral objects such as small dielectric spherical particles that experience two kinds of forces: namely, the scattering force and the gradient force [et al. [Saccharomyces cerevisiae cells [nt force ,130. Thent force . Single nt force ,135,136 nt force  used OT nt force . Thoumin [et al.  were ablae cells ,135,138 Centrifugation Assay. Centrifugation assay is one of the frequently used techniques to measure cells adhesion strength due to their simplicity and the wide availability of equipment in most laboratories. Cells will be seeded in a multiwell plate and undergo treatments by culturing (cell culturing is similar to wash assay) before being spun for the cell detachment process. During the spinning, cells will experience a body force acting in the direction normal to the bottom of the plate that pulls them away from the surface [ surface . To asse surface ,140, qua surface , or by u surface . In manyet al. [HT 1080 cells compared to the cells binding to ECM proteins. García et al. [et al. [HT-1080, NIH3T3 and MC3T3-E1) on different concentrations of collagen or fibronectin. Koo et al. [This method was used by Channavajjala et al.  to undera et al. ,144,145  [et al. ,147 demoo et al.  used theSpinning Disk. The spinning disk technique utilizes shear stress generated from a rotating disk device. Cells are first seeded on circular glass coverslipsor on the surface of a disk . These disks are later fixed onto a rotating device, that is placed inside a chamber filled with buffer solution [MC3T3-E1 cells on RGD peptides on self-assembled monolayers [et al. [et al. observed the effect of transformed oncogene v-src on the adhesion strength of chick embryo fibroblasts [et al. [IGF-I). The effect of surface charges on different substrates has also been studied using HT-1080 cells following fibronectin coating [et al. [et al. [IMR-90 human fibroblasts adhered to fibronectin-coated glass using the spinning disk technique.solution ,148. Thesolution  or automsolution . The spisolution ,151, humsolution ,153, andnolayers . Lee et  [et al.  used thi [et al. ,155. Boeroblasts  and humaroblasts  to under [et al.  investig coating . Reuteli [et al.  were ablFlow Chamber. There are two types of flow chambers used for adhesion strength measurement: the radial and parallel flow chambers. The radial flow chamber methods involve flowing fluid in a chamber over adhered cells on a stationary substrate where a wide range of radially dependent shear stresses are applied [ applied  to detac applied . The rad applied . The lin applied .et al. [et al. [HSMCs) initial attachment strength and migration speed on a range of fibronectin and collagen type IV concentration. Their finding’s suggested that cell-substrate initial attachment strength is a central variable governing cell migration speed and the cell’s maximal migration occurs at an intermediate level of cell-substrate adhesiveness [3T3 fibroblasts on the fibronectin-coated of self-assembled monolayers on glass surfaces [MC3T3-E1 cells to multilayer polyallylamine hydrochloride (PAH) heparin films was analyzed in order to evaluate biocompatibility of various film chemistries [et al. [et al. [Escherichia coli K12-D21 in the in the mid-exponential and stationary growth phases under flow conditions. Chinese hamster ovary cells (CHO) were used to explore the adhesion behavior towards different substrates with different treatments [The radial flow chamber technique has been used by Cozens-Roberts et al.  to inves [et al.  studied siveness . A numbesurfaces ,166,167.mistries . Rezania [et al. ,172,173  [et al. ,170,171  [et al.  showed teatments .The parallel plate flow chamber consists of a bottom plate and an upper plate separated by a distance of the channel’s height to form a rectangular flow channel. Cells are grown on a coverslip and positioned in the flow chamber, constructed by sandwiching a thin rubber gasket between two plates and mounted on a microscope to allow direct observation of the cells during experiments . The floet al. [l-lactide (PLL) films [et al. [VCAM-1) transduced human endothelial cells under physiological flow conditions. Palange et al. [The parallel flow technique was first introduced to study endothelial cell adhesion ,179 and et al.  modifiedet al. ,187. TheL) films ,189, polL) films , polyethL) films , and numL) films ,193,194. [et al.  were able et al.  investigMicrofluidics. Microfluidic lab-on-a-chip technologies represent a revolution of the flow chamber in laboratory experimentation, bringing the benefit of miniaturization, integration, and automation to many research-based industries. These greatly reduce the size of the devices and make many portable instruments affordable with quick data read-outs. The use of small sample volumes leads to greater efficiency of chemical reagents, straightforward construction and operation processes, and low production costs per device, thereby allowing for disposability and fast sampling times. The ability for real-time observation makes microfluidics bring high promises for cell adhesion studies. In recent years, cell adhesion studies have been carried out in a miniature form of the traditional parallel plate flow chambers as discussed above, using flow in rectangular microchannels to apply shear stresses to cells. These devices are typically constructed from optically transparent PDMS bonded to glass using the soft lithography rapid prototyping process that allows many nearly identical devices to be manufactured in a short amount of time [ of time . The opt of time . Small d of time .et al. [NIH/3T3 mouse fibroblast and bovine aeortic endothelial cells) and materials [et al. [et al. [NIH3T3 fibroblast cells that had been allowed to adhere for 24 h on the collagen and fibronectin coatings on glass [A microfluidic device consisting of eight parallel channels has been used to assess the effect of varying collagen and fibronectin concentrations on the adhesion strength of endothelial cells . A serieet al.  used a materials . Recentl [et al.  integrat [et al.  have upgon glass . In thisSingle cell approaches allow for precise measurements of the separation of the cell from the substrate. Specialized equipment, which is bulky and expensive, is often required for manipulation and alignment of the probe and testing can be time-intensive. The single cell adhesion measurement approach provides more precise measurement of the individual cell when compared to the population cell approach. The single cell measurement approach allows the system to image biomolecules at nanometer-scale resolution, to have a dynamic range of forces able to be applied to cells, and to process samples in their physiological medium and aqueous buffer . It has Studying human diseases from a biomechanical perspective can lead to a better understanding of the pathophysiology and pathogenesis of a variety of illnesses because changes occurring at the molecular level will affect, and can be correlated to, changes at the macroscopic level. Research on biomechanics at the cellular and molecular levels not only leads to a better elucidation of the mechanisms behind disease progression, it can also lead to new methods for early disease detection, thus providing important knowledge in the fight and treatments against the diseases. Sickle cell disease (SCD) could be characterized by observing the red blood cells’ (RBC) adhesiveness and deformability ,204,205.Cell adhesion is a very important process in the human biological system. Studying both cell attachment and detachment events provides essential knowledge in understanding many important functional processes in the human body, which lead us to find the causes and problems that trigger certain diseases and thus develop the strategy for curing and improving them. Many different techniques and adhesion assays have been developed to study cell adhesion applicable to a wide range of fields. Every method is unique and was developed for specific important and independent purposes, which makes them difficult to compare in finding the best method applicable for cell adhesion studies. Choosing an appropriate technique is highly dependent on the purpose of the information that a person desires to obtain. Both single cell and population studies are equally important and required to fully understand how cells behave and function in the human system.


Exemplified by cancer cells' preference for glycolysis, for example, the Warburg effect, altered metabolism in tumorigenesis has emerged as an important aspect of cancer in the past 10–20 years. Whether due to changes in regulatory tumor suppressors/oncogenes or by acting as metabolic oncogenes themselves, enzymes involved in the complex network of metabolic pathways are being studied to understand their role and assess their utility as therapeutic targets. Conversion of glycolytic intermediate 3-phosphoglycerate into phosphohydroxypyruvate by the enzyme phosphoglycerate dehydrogenase (PHGDH)—a rate-limiting step in the conversion of 3-phosphoglycerate to serine—represents one such mechanism. Forgotten since classic animal studies in the 1980s, the role of PHGDH as a potential therapeutic target and putative metabolic oncogene has recently reemerged following publication of two prominent papers near-simultaneously in 2011. Since that time, numerous studies and a host of metabolic explanations have been put forward in an attempt to understand the results observed. In this paper, I review the historic progression of our understanding of the role of PHGDH in cancer from the early work by Snell through its reemergence and rise to prominence, culminating in an assessment of subsequent work and what it means for the future of PHGDH. As a genetic disease, cancer is primarily caused by mutations in oncogenes and tumor suppressors, which serve to control tissue homeostasis . Altered in vitro and in vivo [The “Warburg effect” named in his honor describes a process wherein cancer cells preferentially use fermentative glycolysis-based glucose metabolism instead of entering the tricarboxylic acid cycle (TCA) and subsequent electron transport chain, even under aerobic conditions –6. The c in vivo .Metabolic reprograming in tumorigenic cells is not limited to deregulation by oncogenes and tumor suppressors but can also result from genomic modifications to the metabolic enzymes themselves, independently contributing to biomass accumulation and proliferative growth –14. ThisOne example is the conversion of glycolytic intermediate 3-phosphoglycerate into phosphohydroxypyruvate by the enzyme phosphoglycerate dehydrogenase (PHGDH)—a rate-limiting step in the conversion of 3-phosphoglycerate to serine Figures  and 2 1. In the In a review of serine metabolism published in 1984, Snell  provides in vivo did not alter the effect [Two years later, Snell and Weber  publishee effect , leadinge effect  would laSome 19 months later in July 1987, Snell et al. [Finally in January 1988, Snell et al.  publishe PHGDH gene. Citing work by Achouri et al. [ PHGDH gene from a rat hepatoma in 1997, Cho et al. [λ ZAP human Jurkat T-cell cDNA library to look for similarity to the known rat PHGDH gene. One 852 bp clone showed partial similarity to the 3′-region and was subsequently used to rescreen the library as a novel probe [ PHGDH sequences and 3D modeling allowed for the assignment of substrate-binding, nucleotide-binding, and regulatory domains. The overall sequence was found to share 94% homology with rat PHGDH [ PHGDH [Following Snell et al.'s final publication in 1988 , the groi et al.  that repo et al.  used 300el probe . Of fiveat PHGDH  and 93%  [ PHGDH .17-Asp sequence known to be involved in binding the adenosine portion of NAD+, showed the highest degree of homology [When the sequence was compared via individual functional domains, the nucleotide-binding domain, containing a consensus Gly-Xaa-Gly-Xaa-Xaa-Gly-Xaahomology . The C-thomology , 30.32P-labeled version of the 2,478 bp PHGDH cDNA probe on a human multiple tissue northern blot assay [Tissue distribution of PHGDH-specific mRNA was then analyzed using a ot assay . Two traot assay . In contot assay  found a ot assay  note thaot assay  may not ot assay . Taken tot assay  suggest ot assay .Km, Vmax, and/or stability. Akin to their analysis of normal tissue, Cho et al. [Cho et al.  then souo et al.  used a no et al. . The higo et al. . On the o et al.  establiso et al. –20 and s Nature and Nature Genetics, the work of Pollari et al. [Studies of human PHGDH began to appear in subsequent years, prior to prominently reemerging in 2011 with the work of Possemato et al.  and Locai et al.  set the i et al.  led to ti et al. . Subsequ in vivo via intracardiac inoculation of immunodeficient mice. Comparison of parental MDA-MD-231 and an enhanced daughter variant with heightened metastatic abilities, MDA-MB-231(SA), revealed genetic aberrations that were highly conserved between the two lines [P < 0.001) in strongly versus weakly metastatic cells, PSAT1 expression was not significant and PSPH failed to correlate with metastatic ability at all [Highlighting the incurable nature and significant morbidity of bone metastatic disease, Pollari et al.  used breP < 0.001) in addition to shorter overall survival time (P = 0.002). Further assessment for “clinically relevant features” in a subset of 251 samples pointed to additional associations between enzymatic PHGDH and PSAT1 expression and several recognized risk features, including estrogen and progesterone receptor negative status, mutated p53, higher tumor grade, heightened expression of the cell proliferation markers PCNA and Ki-67, and higher levels of ERBB2 [Pollari et al.  concludeof ERBB2 . Nature. With the stated aim of identifying metabolic genes required for tumorigenesis, Possemato et al. [In August 2011, Possemato et al.  publisheo et al.  cross-reo et al.  construco et al. . PHGDH exists in a region of chromosome 1p commonly amplified in several types of cancer, including cancers of the breast and skin (melanoma) [ in vivo screen also decreased PHGDH protein expression; two of differing knockdown efficacies inhibited tumor growth [Consultation of cancerous somatic genome-wide copy number alterations reported in the work of Beroukhim et al.  in an efelanoma) . None ofr growth . PHGDH had the most significantly elevated expression in estrogen receptor-negative breast cancer cells. Among 82 human breast tumor samples assessed in an immunohistochemical assay (not discussed in this review) PHGDH protein levels significantly correlated with estrogen receptor-negative status [The initial results of Possemato et al.  corrobore status . In whate status  conclusie status . PHGDH had 8–12-fold higher PHGDH protein expression relative to nontransformed cell lines (lacking gene-based amplification). Where the subsidiary analysis becomes interesting is in its secondary observation that PHGDH protein levels were elevated in two estrogen receptor-negative cell lines, lacking a PHGDH copy number gain [Turning to yet another line of evidence, the letter by Possemato et al.  continueber gain —a findinTo better understand the metabolic consequences associated with such increased PHGDH expression, Possemato et al.  used metIn their penultimate experiment, Possemato et al.  sought tSuch seemingly contradictory results with respect to serine flux presented a conundrum for Possemato et al. . On the 13C-glutamine further revealed that absolute flux from glutamine to aKG and to other TCA intermediates was significantly reduced in cells with RNAi-mediated suppression of PHGDH [Thus, in their final experiment for the paper, the researchers sought to test the notion that PHGDH, PSAT1, and PSPH reactions may produce metabolites beyond serine critical for cell proliferation . They hyof PHGDH . In cellof PHGDH . Based oof PHGDH  believe  Nature Genetics addressing PHGDH expression in melanoma cells, Locasale et al. [13C-glucose using targeted chromatography and mass spectrometry in HEK293T cells. Labeled glucose was incorporated into 13 metabolites across multiple pathways over a 30 min span [13C-phosphoserine labeling paralleled 13C-serine labeling—data further corroborated by NMR experiments which indicated that a “substantial fraction” of glucose is diverted from 3-phosphoglycerate toward the conversion of serine and glycine in these cells. To measure the total amount of glucose-derived serine being made, Locasale et al. [13C-glucose and measured metabolites in cell extracts using targeted chromatography and mass spectrometry. Labeled serine accounted for about one-half with corresponding amounts of glucose incorporation detected in subsequent nucleotide and nucleoside intermediate formation. Expression of PHGDH was verified by Western blot; Locasale et al. [In their nearly concurrent publication ine et al.  challenge et al. , suggeste et al.  began thmin span . Importae et al.  culturede et al.  reported PHGDH gene. To that end, they identified PHGDH in the work of Slamon et al. [ PHGDH was found in a region of chromosome 1p (1p12) known to exhibit recurring copy number gains in 16% of all cancers [ PHGDH revealed localized amplification within the coding region of the gene. In an effort to verify these findings, the researchers examined focal copy number gain using fluorescence in situ hybridization (FISH) with an esophageal squamous cell carcinoma line (T.T. cells) known to contain amplification of PHGDH. Stable PHGDH knockdown using shRNA reduced the proliferation rate. Moving on to test whether the decreased proliferation was due to reduced ability to utilize the serine biosynthetic pathway, Locasale et al. [Indication of selective glucose diversion led to the notion that there may be a context in which pressure exists for tumors to increase PHGDH activity ; much lin et al.  and note cancers . Furthere et al.  generate PHGDH amplification results and noted that since amplification in a single tumor type was most commonly found in melanoma, it may be of use to consider PHGDH expression and copy number gain in human melanoma tissue samples. To that end, they used immunohistochemistry to measure PHGDH expression and found that high expression (defined by an IHC score > 1) was observed in 21% of samples. Corresponding copy number gains were detected with FISH in a subsample of 21 out of 42 tumors assayed. To investigate whether melanoma cell lines containing PHGDH amplification would be sensitive to decreased expression of PHGDH, cell lines with and without copy number gains were assessed with a methodology similar to that of the studies conducted by Possemato et al. [Echoing the need for human data first espoused by Snell  and Cho o et al.  took to P < 0.0001) with several clinical parameters. In an effort to validate and expand these results, Locasale et al. [P = 0.002) and basal subtypes (P = 0.004) but did not associate with general parameters such as metastasis (as was previously reported) or with tumor size. Consistent with a reliance of a subset of breast cancers on PHGDH, protein expression was required for growth in a panel of three  breast cancer cell lines (including the BT-20 cell line that carries amplification). Reduced PHGDH expression decreased phosphoserine levels in PHGDH amplified BT-20 cells, while nontumorigenic breast cancer epithelial MCF-10a cells did not require PHGDH for growth, exhibit alterations in glycolysis on shRNA knockdown of PHGDH, or show detectable labeling of phosphoserine from glucose [However, while copy number gain offers one mechanism to divert flux into serine biosynthesis, Locasale et al.  also note et al.  assessed glucose . PHGDH on chromosome 1p12 is amplified in some 6% of breast cancers and 40% of melanomas. Beyond genomic amplification, a larger fraction of tumors were found to have elevated PHGDH protein levels, including 70% of estrogen receptor-negative breast cancers. High PHGDH expression (with or without genomic amplification) was associated with dependence on the enzyme for growth either via serine utilization or a hypothesized benefit of aKG to the TCA—a phenomenon that Deberardinis [Given the importance granted to these two papers and the dense nature of the results that they report, it seems prudent to take a moment to review. Both studies published in mid-2011 report that the gene encoding PHGDH is amplified in a significant subset of human tumors and underscore that diversion of glycolytic intermediates into the serine biosynthetic pathway may contribute to tumorigenesis in cancer cells , 21, 34.rardinis  and Setorardinis , 21, 34. in vitro and in vivo. In nude mice injected with stable, PHGDH shRNA-silenced glioma cells, overall survival was prolonged relative to mice injected with wild-type cells. The oncogenic transcription factor FOXM1 was also downregulated in PHGDH shRNA-silenced glioma cells. Using liquid chromatography/liquid chromatography-mass spectrometry, Liu et al. [In a series of papers that followed Liu et al. , Jing etu et al.  identifiu et al.  and, in u et al. .P < 0.05) [P < 0.05)—both of which also associated with tumor progression, stage, and size (P < 0.05) [P = 0.029). On the whole, protein expression of PHGDH tended to be high in patients classified as mixed or basal-like subtype and low in patients classified as immune-related, molecular apocrine, or null subtype but was not statistically different when considered across all six TNBC types (P = 0.070). Among mixed subtype cases, 89.3% showed partial expression of basal markers in their mix [The work of Jing et al.  follows  < 0.05) . Express < 0.05) . Finally < 0.05)  publishe < 0.05)  with est < 0.05) ). Using  < 0.05)  assembleheir mix .Historic progression aside, the question remains “What do these findings actually mean for the utility of PHGDH in cancer cells?” The answer is not clear, rendering PHGDH a fascinating and, at first glance, unintuitive metabolic target in cancer . Known tde novo production of serine and, by way of serine hydroxymethyltransferase, glycine from which multiple amino and nucleic acids can be made. What is interesting about this aspect of the pathway is that cells grown in standard in vitro culture conditions consisting of abundant extracellular serine have been found to be sensitive to PHGDH knockdown [ de novo synthesis of an otherwise nonessential amino acid preferentially occurs such that in its absence cell proliferation suffers or does not occur. This notion of dependency or “addiction” to a “flexible flux” [Considered in more detail, the first and perhaps most readily apparent benefit involves the products of serine biosynthesis itself—nockdown . Perhapsnockdown  suggest,le flux” , 34 lead c-Myc with serine hydroxymethyltransferase overexpression leading to stimulated proliferation in c-Myc-deficient cells. At the same time, both serine and glycine are abundant in the plasma, so as noted previously by Mullarky et al. [Tying into the conversion of serine to glycine by the enzyme serine hydroxymethyltransferase, the second notion stems from recognition that this conversion provides a major source of methyl groups for the one carbon pools required for biosynthesis and DNA methylation , 6. By cy et al. .Perhaps the role of aKG used by Possemato et al.  to expla3−) and the reversible transamination of aspartate to form oxaloacetate (including generation of glutamate and depletion of aKG) coupled with the oxidation of glutamate back to aKG (glutamate + NAD+ + H2O → NH4+ + aKG + NADH + H+) or the conversion of propionyl-CoA to succinyl-CoA (propionyl-CoA + ATP + HCO3−  → succinyl-CoA + ADP + Pi) in the β-oxidation of fatty acids [The suggestion that aKG synthesized from serine is critical for tumor growth is not easy to understand from a metabolic point of view given the numerous potential physiological sources of the compound . It is gty acids . Neverthty acids  acknowle+. How this is accomplished remains unclear. Mullarky et al. [+ on mitochondrial electron transport chain complex II, may provide a means. In such a situation, the purported pathway could provide an additional benefit to cancer cells by enabling mitochondrial ATP synthesis to occur with less production of reactive oxygen species, passing the electrons from NADH to complex II instead of conventional passage to complex I [Allowing for a greater amount of speculation, Mullarky et al.  contend y et al.  contend omplex I .Finally, Mullen and DeBerardinis  succinct PHGDH amplification likely stems from “a combinatorial effect of pathway flux toward biomass production, changes in redox status, energy metabolism, and possibly some signaling functions” in a manner that “likely varies based on environmental factors, tissue of origin, and cooperating oncogenic mutations” [Across all of these theories, the one thing that becomes collectively clear is that although glycolysis, the TCA cycle, and glutamine metabolism are central to the functioning of normal, and at least to some extent cancerous, cells, they do not act alone . They artations” . If the Moving forward, the challenge will be to determine whether or not cancer cells expressing elevated levels of PHGDH require additional serine for growth. Evidence presented throughout this paper speaks to a complicated and uncertain result, for, as we have seen in the work of Locasale et al. , higher ζ [ζ function in mice results in increased tumorigenesis and heightened expression of PHGDH as well as PSAT1. Mechanistically, crystal structures of PHGDH dimers reveal phosphorylation sites at Ser55, Thr57, and Thr78 thought to be highly conserved among humans, rats, monkeys, and mice [Ongoing work in the field has begun to consider intersecting pathways such as reported correlations between serine biosynthesis and p73 expression in human lung adenocarcinomas  as well ζ . Recent ζ  providesand mice . In prosand mice , and in and mice . Additioand mice  and Al-Dand mice .In terms of using PHGDH as a potential therapeutic target, one would need to establish a significant difference between the requirement of the enzyme's activity in cancer and in normally proliferating cells . Over th


Keratitis is an inflammatory condition, characterized by involvement of corneal tissues. Most recurrent challenge of keratitis is infection. Bacteria, virus, fungus and parasitic organism have potential to cause infection. TLR are an important class of protein which has a major role in innate immune response to combat with pathogens. In last past years, extensive research efforts have provided considerable abundance information regarding the role of TLR in various types of keratitis. This paper focuses to review the recent literature illustrating amoebic, bacterial, fungal and viral keratitis associated with Toll-like receptor molecules and summarize existing thoughts on pathogenesis and treatment besides future probabilities for prevention against TLR-associated keratitis. Toxoplasma gondii acts as a first ligand for TLR11 that has been described as an important clue regarding the gene expression studies on mice but not on human. MyD88 plays an essential role in TLR signaling and is involved with TLR expression in eye  is a painful and most serious corneal infection, caused by strain of protozoan—El-Sibae . DiagnosPseudomanas aeruginosa, Staphyloccus aureus and Streptococcus pneumonia. Gram-negative bacteria, Pseudomanas aeruginosa, are a major cause of bacterial keratitis in Unites States and have been found as a risk factor in contact lens wearer  and adenovirus keratitis. HSV is an enveloped double-stranded DNA virus and considerably causes ocular infection. Epidemic studies of ocular HSV infection showed that HSV-1 is a prominent cause of viral keratitis  demonstrated that corneal blindness caused by microbial keratitis is emerging as a main cause of visual impairment after cataract and glaucoma  production in cornea of Wistar rat using inhibitors like PDTC and U0126. Similar findings have been obtained by Ren et al. (Acanthamoeba.Ren and Wu  investign et al.  in humanPseudomonas aeruginosa endotoxin-induced keratitis in BALB/c, C3H/HeN mice. They measure stromal thickness, haziness and neutrophil recruitment by in vivo scanning confocal microscopy and immune histochemistry, respectively. Saint Andre et al. (Wolbachia play an important role in corneal inflammation induced by bacterial lipopeptides that activate TLR2/TLR6/MyD88 signaling in the cornea. Zhang et al. (Pseudomonas aeuroginsa infection. They determined IKβ-α phosphorylation and degradation, expression of IL-6, IL-8 in mRNA, and secretion using Western blotting, RT-PCR and ELISA, respectively. Johnson et al. (Staphyloccousaureus or synthetic lipopeptide Pam3cys. Huang et al. (Pseudomonas aeruginosa. Carlson et al. (Pseudomonas keratitis. Huang et al. (Pseudomonas aeruginosa but have higher potential for systemic infection. Ito and Hamerman (Pseudomaonas aeruginosa infection in mice. Huang et al. (Pseudomonas aeruginosa. Hayashi et al. (It has been seen in various researches that modulation in TLR signaling occurs in corneal epithelium cells in several ways after infection with bacterial pathogens, responsible for bacterial keratitis. Studies shows a role of different types of TLR and mechanism associated with TLR-linked bacterial keratitis. Khatri et al.  determine et al.  show thag et al.  examinedn et al.  demonstrn et al.  exploredg et al.  showed tn et al.  demonstrn et al.  examinedn et al.  demonstrn et al.  show thag et al.  show thaHamerman  observedHamerman  displayeg et al.  showed ti et al.  revealedi et al.  demonstri et al.  demonstrKariko et al.  reportedFusariumsolani fungus in BALB/c mice. Gao and Wu (Aspergillus fumigates and Fusarium solani. Jin et al. (Fusarium solani. Yuan and Wilhelmus (Candida albican infection using BALB/c and C57BL/6 mice. Sun et al. (Fusarium keratitis. Hua et al. (Fusarium solani keratitis in BALB/c mice and TLR4 also involved in controlling fungal infection during Fusarium oxysporum keratitis in C57BL/6 mice. Tarabishy et al. (−/− and TLR4−/− mice, but not TLR2−/− mice, have an initially increased F. oxysporum burden because of reduced fungal clearance in the cornea. In C. albicans keratitis, TLR-knockout mice are helpful to determine the effect of TLR2 and 4 in experiment yet, the severity of fungal keratitis in murine mutant strains was similar to that in wild-type control mice, more fungi were recovered after 3 days of infection from TLR2−/− than from TLR4−/− mouse corneas. The results of this study were similar to another study done by Villamon et al. (Oropharyngeal candidiasis, the level of TLR2 and 4 increased after fungal infection in mice. MyD88 is also essential for TLR2 and TLR4 signaling, yet the role of TLR2 and TLR4 is not clear in the development of corneal opacification; TLR4−/− mice have an impaired ability to clear the infection so TLR4−/− is important in eradicating fungal infection. Gao and Wu (Aspergillus fumigates and Fusarium solani. Bellocchio et al. (A. fumigatus and to the pathogenic yeasts C. albicans and Cryptococcus neoformans (Table Hu et al.  observedo and Wu  and Zhaoo and Wu  elucidatn et al.  observedilhelmus  showed tn et al.  determina et al.  and Taraa et al.  showed ty et al.  displayen et al. . Zhang en et al.  showed to and Wu , Zhao ano and Wu , Guo et o and Wu , Guo ando and Wu , Zhao eto and Wu  and Hu eo and Wu  showed to et al.  and Meieo et al.  elucidatns Table .Table 1LStaphylococcus. Huang et al. (Pseudomonas aeruginosa keratitis in BALB/c mice. Zhang et al. (Pseudomonas aeruginosa infection in human corneal epithelium cell. Guo et al. (Apergillus fumigatus keratitis by suppressing corneal inflammation. Wilhelmus (Acanthamoeba keratitis usually affect contact lens wearer and to prevent this problem improvement in disinfecting solution has been done because Verani et al. (Acanthamoeba sometimes anti-Acanthamoebic efficacy of solution was not sufficient. Hara et al. (Sun and Pearlman  investigg et al.  elucidatg et al.  used anto et al.  determinilhelmus  showed ti et al.  shows tha et al.  proposeda et al.  demonstrIn above studies, animals like rabbit and mice have been mostly used for studying different types of keratitis. There has been found several disadvantages of using animal models because of physiological differences between human and animal eye like corneal size, corneal thickness, arrangement of corneal collagen (Hayes et al. In many research broad spectrum antibiotics like vidarabine, trifluridine, acyclovir or gancyclovir linked with several side effects/risks because broad spectrum antibiotics can disturb normal flora in eye and drugs can be associated with drug resistance by pathogenic bacteria.Improvement in corneal cell culture models would be useful in pathogenesis of ocular diseases because it can reduce risk associated with killing of animals. Some researchers use primary cell culture of corneal epithelial cells and cell lines with long lifespan as in vitro models for ocular toxicology studies and to explore human corneal epithelial cell biology but it is difficult to cultivate primary human corneal epithelium because of paucity of accessible tissue So, SV 40-immortalized HCEC lines with properties that have resemblance with normal human corneal epithelial cells can be used in pathogenesis of keratitis problem. It also has been observed that artificial corneal epithelium cell under serum-free conditions can act as better model than normal corneal epithelium cell for ocular surface studies. These cell cultures are useful for studying gene regulation and tissue development studies.Understanding complex mechanisms associated with TLR-linked corneal inflammation will be helpful to device new therapeutic approach to modulate immune responses associated with TLR. Since some studies show that understanding of molecular pathways of TLR and RIG/Mda 5, which activate immune response against antiviral infection, will lead to novel approach for treating antiviral infection. Small molecules have been used for better understanding of molecular basis of infective keratitis. Toll like receptor namely TLR7 and TLR 8 can independently mediate recognition of small compounds like “imidazoquinoline R-848” suggest possible redundancy in these receptors. Understanding the function and biology of the corneal LPS receptor complex may lead to novel therapies for the management of ocular Gram-negative bacterial infections has been seen.Micavibrioaeruginosavorous and Bdellovibriobaceriovorous may be susceptible to attack pathogenic MDR bacteria. These good bacteria can combat with bad bacteria.RNAi pathway is often providing good advantage to investigate function of genes in cell culture studies according to previous researches. RNAi is fast, uncomplicated and reliable effort to repress targeted genes expression. Therapeutic potential of RNAi has been revealed in several diseases like viral infection, hepatitis and ocular neovascularization. siRNA technique provides great advantage due to facile delivery of siRNA on cornea. It also has major applications in gene knockdown, functional genomics, medicinal field and biotechnology field studies. RNAi drugs show better response than antisense RNA molecules and antibody-based drugs. RNAi may be more effective than antisense RNA in human cancer cell lines. To deal with antibiotic resistance bacteria problem, some bacteria can be used to combat with drug-resistance bacteria. Predator bacteria like


It is now well established that psychosocial factors can adversely impact the outcome of spine surgery. This article discusses in detail one such recently-identified “risk” factor: demoralization. Several studies conducted by the author indicate that demoralization, an emotional construct distinct from depression, is associated with poorer pain reduction, less functional improvement and decreased satisfaction among spine surgery patients. However, there are indications that the adverse impact of risk factors such as demoralization can be mitigated by psychosocial “maximizing” factors—characteristics that propel the patient towards positive surgical results. One of these maximizing factors, patient activation, is discussed in depth. The patient activation measure (PAM), an inventory assessing the extent to which patients are active and engaged in their health care, is associated not only with improved spine surgery results, but with better outcomes across a broad range of medical conditions. Other maximizing factors are discussed in this article. The author concludes that the past research focus on psychosocial risk factors has limited the value of presurgical psychological screening, and that future research, as well as clinical assessment, should recognize that the importance of evaluating patients’ strengths as well as their vulnerabilities. For example, Rajaee et al.  examinin [et al.  who founet al. [et al. [et al. [Spine surgery can be, and often is, quite effective. For example, Weinstein et al.  in the S [et al.  found th [et al. ).et al. [et al. [On the other hand, it is well established that spine surgery not infrequently fails to provide pain relief and improved functional ability. For example, a recent analysis of discectomy patients found 28% had unfavorable outcomes , with a et al.  found th [et al.  for a syet al. [et al. [et al. [et al. [A large and growing body of research is finding that psychosocial risk factors can contribute significantly to the variability in spine surgery outcome , and ot [et al. ; Trief,  [et al. ; Voohies [et al. ; Chiacha [et al. ) demonst [et al. ) patientet al. [2 = 0.88, and internal consistency (r2 ranging from 0.87 to 0.93 depending on the population tested), with no significant differences between average scores of men and women , was based on the use of the original Minnesota Multiphasic Personality Inventory (Hathaway and McKinley ) or its et al.  found thn-Porath , pp. 24–et al. [Our research  has fouet al. ) and pooet al. ). Furtheet al.  T-scoreset al.  by our gElevated scores on scale RCd have also been found to be associated with poorer conservative treatment outcomes in chronic low back pain. Tarescavage, Scheman and Ben-Porath  examininet al. [et al. [et al. [A substantial literature exists on demoralization, especially in the context of other chronic medical conditions. Most of these studies use instruments other than the MMPI-2-RF, including the Diagnostic Criteria for Psychosomatic Research  or the  [et al. ). A rece [et al. ) found ti.e., inability to experience pleasure . Demoragueiredo ). Severagueiredo  examinin [et al.  found am [et al. ; Adogwa  [et al. ), which  [et al. ), demoraThe feelings of ineffectiveness, helplessness and the sense of giving up that comprise the core of demoralization stand in sharp contrast to the behaviors and general health orientation that are associated with positive health outcomes. In order to achieve and maintain good health, individuals must be able take control over diet and exercise and seek out health information. Individuals also need to recognize when illness occurs, and be able to communicate with health care providers. They need to work with their physicians on plans to overcome or mitigate illness, and have the fortitude to follow through on these plans. Such an effective health orientation is captured by the Patient Activation Measure .1c, and testing for low-density lipoprotein cholesterol, among others . Belief that taking an active role in health is important; (2). Having the confidence and knowledge to take action; (3). Taking health-related action; (4). Staying the course under stress. In the original studies, PAM scores correlated significantly with the use of a glucose journal in diabetes, with following a low fat diet in patients with high cholesterol, with routinely exercising for patients with arthritis, and for seeking out information from health care providers. Further studies have found the PAM correlates significantly with both health outcomes and health care utilization. For example, in diabetics PAM scores predicted testing for, and control of Hemoglobin And Jones ). In an nd Jones ).Two previous studies have examined the role of patient activation in spine surgery patients. Skolasky, Mackenzie, Wegener and Riley  examinedet al. Unpublished data [2 = 0.33, p < 0.01 ), reduction of negative affect as assessed by change scores on Likert-type emotion ratings , and with patient satisfaction  at an average of about 5 months post-op.We have been examining the relationship of the PAM to the outcome of spine surgery. Thus far, we  have giviz, the assessment of patient characteristics that may militate towards improved outcomes. Certainly, my own research  as well [et al. ; denBoer [et al. ), which  [et al. ) have foet al. [Other potential “maximizing factors” warrant exploration. Consider, for example, a patient who has a high level of family problems . Such a patient may simultaneously have a strong social support system outside the family, or even be satisfied with the level of support received by family members, despite the problems that exist within the family. Social support has been found to be an important predictor of improved health outcomes. For example, Mutran, Reitez, Mossey and Fernandez , examiniet al.  found thet al. ). Thus, et al. [et al. [et al. [A third potential characteristic that may help to maximize spine surgery results revolves around expectations for the outcome of spine surgery. Spine surgery has three major goals: reduce pain; improve functional ability; and correct the underlying physical pathology responsible for the pain and functional deficits. The extent to which these three goals are achieved, however, varies widely. Some patients coming to surgery expect total pain relief and a complete return to pre-morbid activity levels, while others may consent to surgery even though their expectation is that minimal improvements will occur. So, it is reasonable to consider whether patient expectations bear a relationship to surgical results. Although the results are not completely consistent, several studies show that greater expectations of improvement assessed pre-operatively are associated with more sanguine surgical results. For example, Yee et al.  examininet al. ) found h [et al.  examinin [et al. ). Thus, --Positive pain coping strategies, such as optimism  and Haret al. );--et al. [Spirituality and forgiveness .Patient activation, social support and positive outcome expectations are but three of a host of psychosocial factors that might potentially be associated with improved spine surgery results. Some other factors that have been found to correlate with improved outcome of treatment for pain, and may militate towards better spine surgery response include: It would be of great value to explore these and other positive factors that may contribute to better spine surgery results.A number of psychosocial risk factors for reduced spine surgery outcome are by now well established. Depression, somatic sensitivity, demoralization, substance abuse, vocational issues such as workers’ compensation and litigation—all these are shown to have strong empirically-derived correlations with diminished results. However, the focus of PPS upon psychosocial risk factors has ignored much of the complexity of each case and provided limited insight into factors that may improve surgical outcomes. Research on patient activation, social support and surgical outcome expectations point to the importance of examining psychosocial “maximizing factors”—those patient characteristics that may mitigate the adverse impact of established risk factors, and may propel the patient towards good surgical response. In order to provide a full and effective picture of each patient’s capacity for achieving reduction in pain and improvement in functional ability, the field of presurgical psychological screening must begin to focus as much on the patient’s strengths as upon his or her vulnerabilities.


Selected papers from the 13th International Conference on Bioinformatics (InCoB2014), July 31-2 August, 2014 in Sydney, Australia have been compiled in this supplement. These range from network analysis and gene regulatory networks to systems level biological analysis, providing the 2014 update to InCoB's computational systems biology research. Sydney, Australia hosted the 13th InCoB , the official conference of the Asia-Pacific Bioinformatics Network (APBioNet) . This inAuthors were offered the BMC track  and PeerJ . The detet al.[et al.[et al.[et al.[The complexity of biological systems of often represented in terms of networks where biomolecules that interact with each other are represented as interaction partners. Characterizing a complex biological network by measuring its topology using 11 parameters and identifying nodes and sub-networks, is computationally challenging. Chin et al. have devet al. have devet al. have anal.[et al. have coml.[et al.. For sysl.[et al. have appet al.[cisMEP database comprising predicted cis-regulatory modules integrated with available epigenetic data, as a first step to support research in this area. Approaching the transcription regulation problem from another angle, Yan and Wang [Understanding transcriptional regulation of genes remains a challenging problem, dependent on the binding of several transcription factors as well as epigenetic changes. Given the sparseness of experimental genome-wide epigenetic profiles, Yang et al. have devand Wang  propose et al.[et al.[A comprehensive understanding of the symbiosis between the human microbiome and the host organism is essential for defining its role in human health and disease. Yang et al. have appl.[et al. have anaThe articles in this supplement span network analysis, regulatory networks as well as systems-level analysis. With ongoing NIH Big Data to Knowledge (DB2K)  and otheThe authors declare that they have no competing interests.SR wrote the introduction. CS and SR (Program Committee Co-chairs) managed the review and editorial processes, respectively. TWT supported the post-acceptance manuscript processing.


Translational bioinformatics is an emerging field that aims to exploit various kinds of biological data for useful knowledge to be translated into clinical practice. However, the flooding of the huge amount of omics data makes it a big challenge to analyze and to interpret these data. Therefore, it is highly demanded to develop new efficient computational methodologies, especially data mining approaches, for translational bioinformatics. Under these circumstances, this special issue aims to present the recent progress on data mining techniques that have been developed for handling the huge amount of biological data arising in translational bioinformatics field.In data mining, one of the most important problems is how to represent the data so that the computational approaches could handle these data appropriately. In this special issue, B. Gan et al. utilized the latent low-rank representation to extract useful signals from noisy gene expression data and then classified tumors with sparse representation classifier and obtained promising results on benchmark datasets. C. Zhao et al. proposed a new feature representation of facial complexion for diagnosis in traditional Chinese medicine and achieved high recognition accuracy. G. Zhang et al. formulated the skin biopsy image annotation as a multi-instance multilabel (MLML) problem and automatically annotated the skin biopsy images with a sparse Bayesian MLML algorithm based on region structures and texture features. Except for feature extraction, feature selection is also very important in data mining. Z. Ji et al. proposed a particle swarm optimization-based feature selection approach to predict syndromes for hepatocellular carcinoma and improved diagnosis accuracy. With the accumulation of various data in translational bioinformatics, it is becoming a challenging task for traditional intelligent approaches to handle and interpret these data; S. Li et al. presented a survey on the recent progress about the hybrid intelligences and their applications in bioinformatics, where the hybrid intelligence is more powerful and robust compared with traditional intelligent approaches.The rapid accumulation of various kinds of biological data requires more powerful statistical approaches to extract useful signals from the huge amount of noisy data. L. Sun et al. built a new pipeline to investigate the DNA methylation profiles in male and female nonagenarians/centenarians and identified some differentially methylated probes between male and female nonagenarians/centenarians, which provide insights into the mechanism of longevity gender gap of human beings. Z. Teng et al. developed a new algorithm to predict protein function based on weighted mapping of domains and GO terms, which outperforms other popular approaches on benchmark datasets. J.-L. Huang et al. presented an online cross-species comparative system to identify conserved and exclusive simple sequence repeats within model species, which can facilitate both evolutionary studies and understanding of gene functions. L. Guo et al. proposed a new approach to identify microRNAs (miRNAs) associated with breast cancer and found that miRNA gene clusters demonstrate consistent deregulation patterns despite their different expression levels, which may provide insights into the regulatory roles of miRNAs in tumors.β to ovarian carcinoma immunoreactive antigen-like protein 2 (OCIAD2) by exploring the pathway bridge, and the resultant pathway explained how TGF β affects the expression of OCIAD2 in cancer microenvironment.Recently, network biology is becoming a promising research field by organizing different kinds of data into a network representation. T. Jacquemin et al. proposed a new approach to identify disease associated protein complexes based on a heterogeneous network that consists of a disease similarity network and a tissue-specific protein-protein interactions network and successfully found disease associated complexes. X. Li et al. proposed a new pipeline to detect symptom-gene associations by integrating multiple data sources and found some potential disease genes. It is known that DNA mutations will affect gene expression. However, it is difficult to know which mutations will affect the gene expression and how the genes are regulated within the biological system. D. Kim et al. developed a novel approach that can both identify the Quantitative Trait Loci and infer the gene regulation network and successfully identified the genes associated with psychiatric disorder. R. Zhang et al. presented a new approach to identify the pathways linking TGF Xing-Ming ZhaoJean X. GaoJose C. Nacher


We genotyped variants using PCR and direct sequencing and evaluated estimated haplotypes of MDR1 variants. The analysis revealed few differences in SNP genotype frequencies between patients and controls, or in clinical parameters among the patients. Genotype distribution of MDR1 SNPs rs1236, rs2677, and rs3435 showed significant (p < 0.05) association with different medication regimes . Some marginal association was detected between ANGPTL4, GPC5, GLCCI1, and NR3C1 variants and different medication regimes, number of relapses, and age of onset. Conclusion. While MDR1 variant genotype distribution associated with different medication regimes, the other analyzed gene variants showed only little or marginal clinical relevance in INS.Polymorphic variants in several molecules involved in the glomerular function and drug metabolism have been implicated in the pathophysiology of pediatric idiopathic nephrotic syndrome (INS), but the results remain inconsistent. We analyzed the association of eleven allelic variants in eight genes Childhood-onset idiopathic nephrotic syndrome (INS) is a common kidney disease in children. It is characterized by minimal glomerular changes in light microscopy and podocyte foot process effacement in electron microscopy. The great majority (80–90%) of INS patients show good responsiveness to steroid treatment, but recurrent episodes occur in at least 70% of the patients. Development of renal failure occurs rarely.Angptl4), glycoprotein, which is upregulated in rats with steroid sensitive proteinuria  in patients who received IS drug medication instead of GCs only.ntal INS , 25. The4 levels . RecentlGPC5) gene and acquired NS  through a genome wide association study and replication analysis. They showed that glypican-5 is localized on podocyte cell surface membranes and that the risk genotype (AA) of the GPC5 SNP rs16946160 was associated with higher expression. In our study, we observed an association between rs16946160 A allele and early disease onset (16 versus 5%) but we did not find an association between this SNP and INS in general. It is, however, notable that none of our patients and only one of the controls carried the AA genotype. Okamoto et al. found the A allele frequency of controls to be 0.168 and dbSNP (http://www.ncbi.nlm.nih.gov/snp/) puts it at 0.161. In our study, it was only 0.08. Thus, it is possible that due to the frequency differences between populations the association between the risk genotype and INS is not visible in our patients.Recently, Okamoto et al.  identifi nNOS gene polymorphism rs2682826, the TT genotype was associated with INS but not with GC responsiveness. NO attenuates many functions in the kidney and all forms of NOS are expressed in the kidney but the role of NO in renal disease is unclear. In our study, we did not find an association of rs2682826 genotypes with INS or with any clinical features of the disease.Alasehirli et al.  found th IL-13 and MIF, whose genetic variants have been associated with NS, were also negative. While Wei et al. [ IL-13 gene correlate with long term outcome of INS, we did not see any association between the analyzed SNP and the number of relapses, response to medication, or any other feature. MIF is counterregulated by glucocorticoids and the rs755622 SNPs have been studied in association with NS. Vivarelli et al. [Our results of the two cytokines,i et al.  reportedMDR-1 gene codes for a membranous P-gp, which is a multidrug transporter expressed in the proximal tubule cells. Certain SNPs in MDR1 gene are believed to affect the expression of the gene or activity of the protein it codes. The common SNP rs1045642 in exon 26 has garnered a lot of attention. It is synonymously variant and it has been suggested that it is not causal itself but linked with another polymorphism or has an effect on DNA structure or RNA stability [tability . Of the tability , 28. Thetability , 12 whiltability , 22. MDR1 SNP rs1045642 CC genotype showed association with higher age of onset (20 versus 0%). Youssef et al. reported similar association for SNPS rs2032582 as well as rs1045642. Other studies did not show this association [We did not find any difference in the Finnish patients and controls in rs2032582 genotypes. Wasilewska et al. , Jafar eociation , 22. MDR1 SNPs showed association with treatment choices, T allele and TT genotype being more common in patients who needed IS drugs compared to those who were only medicated with GCs, which indicates that T and TT are associated with more complicated form of the disease. Surprisingly, only rs1045642 showed significant association between genotype distribution and GC responsiveness , although it must be noted that only ten of our patients were not responsive to GCs; this small cohort size may affect these results.In our study, all three MDR1 variants. In previous studies, Wasilewska et al. [We carried out haplotype analysis of the threea et al.  reporteda et al.  and Yousa et al. , althouga et al.  reached a et al. , 22, 30.a et al.  found haNR3C1 codes for glucocorticoid receptor (GR) that can affect the regulation of many biological functions, including responsiveness to GC, and its functional variability may play a role in the therapeutic response to GC. In this study, we analyzed NR3C1 SNP rs41423247 and curiously found that patients with more than five relapses carried more frequently heterozygous GC genotype than those with less than five relapses (68 versus 33%). The amount of both CC and GG homozygotes was diminished in these frequent relapsers. The allele distribution between groups with over and under five relapses showed no difference. In some previous studies, G allele  has been associated with increased GC sensitivity [sitivity , 32 whil GLCCI1. Tantisira et al. [An interesting new gene in the context of INS isa et al.  first sha et al.  showed ta et al.  looked tThe studied genetic variants have little role in the course of NS in Finnish patients. A notable exception to this is MDR1 SNPs whose genotype and allele distribution show significant association to different medication regimes. The genetic background to GC sensitivity is very heterogenic and varies between ethnic groups, which may have to be considered when drawing up treatment strategies for individual patients. More work needs to be done to discover other contributing molecules before the genetics of steroid responsiveness in NS can be understood.


It was treated with 6% lime and incubated for 1 week under room temperature in 2 kg sealed polythene bags and was evaluated for proximate composition after incubation. Different isonitrogenous complete diets containing 0-50% of lime treated olive cake on ADF replacement basis were formulated as per the requirement of adult male goats. In ADF replacement, fiber and concentrate sources were replaced by lime treated olive cake by replacing the 0-50% ADF percentage of the total 40% ADF value of complete feed. The formulated complete diets were tested for in vitro dry matter degradation (IVDMD) values were comparable at all replacement levels. However, a point of inflection was observed at 40% ADF replacement level, which was supported by truly degradable organic matter (TDOM), microbial biomass production (MBP), efficiency of MBP and partitioning factor values (PF).Treatment of olive cake with 6% slaked lime increased availability of cellulose and alleviated digestibility depression caused by high ether extract percentage. Organic matter, nitrogen free extract, ADF and neutral detergent fiber were significantly lowered by lime treatment of olive cake. The cornell net carbohydrate and protein system analysis showed that non-degradable protein represented by acid detergent insoluble nitrogen (ADIN) was 21.71% whereas the non-available protein represented by neutral detergent insoluble nitrogen (NDIN) was 38.86% in crude olive cake. The in vitro studies that Indian olive cake can be included in complete feed at 30% level  for feeding in small ruminants without compromising in vitro degradability of the feed.In our study, we concluded that there is comparable difference in composition of Indian olive cake when compared with European olive cake. The most important finding was that about 78% of nitrogen present in Indian olive cake is available to animal in contrary to that of European olive cake. We concluded from  The shortage and increasing cost of conventional feed ingredients have driven the attention of research towards utilization of non-conventional feedstuffs in livestock ration. The use of non-conventional feedstuffs minimizes the competition of livestock with humans for conventional food grains and reduces the cost of animal production -3.Olea europaea L.) oil industry by-products are promising unconventional feedstuffs ) × 100/TDOMPF = TDOM/net gas volumeGeneralized linear model analysis of variance procedure  was usedThe proximate composition and fiber fractions of olive cake and lime-treated olive cake used in this study are shown in The ADIN  was 21.71% whereas the NDIN  was 38.86%. Around 78% of nitrogen is likely to be available to the ruminant. Fraction A which gets instantaneously solubilized in rumen and is cent percent digested in the intestine was 33.69%. The protein fractions likely to be degraded in the rumen were 56% and undegradable dietary nitrogen component was 22.29% .The IVDMD values were comparable (p>0.05) between different inclusion levels. The IVDMD values varied from 48.75% to 56.25%. The TDOM (mg/200 mg DM) values varied from 97.50 to 112 with significant (p<0.01) difference among various replacement levels. The 40% ADF replacement level differed significantly (p<0.01) in TDOM from all other levels except 30% and 35% replacement level. The gas volume varied from 24 to 33 ml/200 mg DM and the levels differed significantly (p<0.01) from each other. The MBP value varied from 33.45 to 48.80 mg/200 mgDM. The MBP value was highest in 40% ADF replacement level and differed significantly (p<0.01) from all other levels. The EMP values varied from 33.42 to 46.41(%TDOM) and differed significantly (p<0.01) in various replacement levels. The values were highest in 40%, 45%, and 50% ADF replacement levels. The PF values varied from 3.31 to 4.12 and the values differed significantly (p<0.01) in various replacement levels. The values were highest and comparable (p>0.05) in 40%, 45% and 50% ADF replacement levels.et al. [et al. [et al [et al. [et al. [et al. [et al. [et al. [et al. [et al [et al [Proximate composition of the olive cake was similar to that reported by Ahmad , Ashraf et al. . CP cont [et al. , Al-Masr [et al. , Vargas-. [et al , Martín  [et al. , Molina- [et al.  and Sade [et al. ; but wer [et al.  and Gul  [et al. . The per [et al. , Al-Masr [et al. , but is  [et al. , Bashir  [et al. , Alhamad [et al. , Luciano. [et al , Awawdeh. [et al  and Vargl [et al .et al., [et al., [et al. [et al. [Chemical composition of olive cake has been shown to be influenced by factors such as geographical origin, procedure of production and processing . Differeet al., , Al-Masret al., , Martín [et al., , Al-Masr[et al.,  and Moli [et al. , which m [et al. .et al. [et al. [et al. [et al. [et al. [et al. [et al. [The NDF and ADF content of the crude olive cake were found to be 80.33 and 62.00%, respectively on DMB. The values were higher to those recorded by Ahmad , Ashraf et al. , Martín  [et al. , Molina- [et al. , Chiofal [et al.  and Rowg [et al. . However [et al.  Abarghoe [et al.  and Al-M [et al. . The var [et al. , Ashraf  [et al.  and Bash [et al. .et al. [Slaked lime treatment was used as per Ashraf  to improet al. . HoweverOlive cake as a feedstuff blurs the demarcating criteria between roughage and concentrates. By standard classifying pattern of feedstuffs, it should be classified as extremely poor quality roughage. However, its higher EE% makes it better concentrate. Although, it is an oil seed cake, it is extremely poor in protein and in contrast, it is almost 50-60% fiber, which makes it an ideal candidate for quality improvement through lime treatment.et al [2 which is slowly degraded amounts to 5.14%. Fraction C which is undegradable was found to be 21.71%. Many previous workers have reported protein degradability of olive cake is poor, owing to the fact that 75-90% of the nitrogen is linked to the ligno-cellulose fraction [The crude olive cake was evaluated for protein fractions as per CNCP system by Sniffen et al . It was fraction -27 therefraction , which win vitro gas production technique as per Menke and Steingass [Ten isonitrogenous complete diets containing variable levels of lime treated olive cake on ADF replacement basis were formulated and tested through teingass .et al. [et al. [et al. [The IVDMD values obtained were compared to select the complete feed with maximum inclusion level of lime treated olive without significant decrease in IVDMD. The IVDMD values obtained were intermediate to the values reported by Vera et al.  and Shab [et al.  but were [et al. . The IVD [et al. .in vitro analysis that olive cake can be included in complete feed at 30% level  for feeding in small ruminants without compromising in vitro degradability.In our study, we concluded that there is a comparable difference in composition of Indian olive cake when compared to European olive cake. The most important finding was that about 78% of nitrogen present in Indian olive cake is available to the animal. We concluded from AI carried out the research work and drafted the manuscript, RKS and AR planned and supervised the research work, BAM and JF helped in conducting lab trial and AI and AR revised the manuscript. All authors read and approved the final manuscript.


Pain is the most common health challenge that drives patients to consult physicians. Pain and inflammation are usually symptoms of many diseases. Inability to perceive sensory pain may lead to shortened life expectancy and yet excessive pain may result in low quality of life. Despite the arrays of therapies available for the treatment of pain, it is still not tamed and the currently available drugs have their drawbacks. The challenges to amelioration of pain and inflammation have become an impetus for research into alternative and complementary medicines for the treatment of these ailments. The roles of phytotherapy and nutritional therapies in the treatment of varied types of ailments cannot be overemphasized. Actually, most of the commonly available therapeutic drugs for pain, such as opioids and nonsteroidal anti-inflammatory drugs (NSAIDs), were derived from phytotherapy. Hence, giving attention to this issue is highly recommended as it provides a medium for knowledge acquisition for improvement of the treatment of the twin ailment-pain and inflammation. Physalis alkekengi var. franchetii and Its Main Constituents” as well as R. Boukhary et al., “Anti-Inflammatory and Antioxidant Activities of Salvia fruticosa: An HPLC Determination of Phenolic Contents.” In another study, I. J. M. Santos et al. reported “Topical Anti-Inflammatory Activity of Oil from Tropidurus hispidus .” A review was accepted for inclusion in the issue on effectiveness of acupuncture for treating sciatica by Z. Qin et al. A. Rauf et al. reported on a potential cyclooxygenase inhibitor named daturaolone while W. Kim et al. evaluated the anti-inflammatory potential of a new Ganghwaljetongyeum (N-GHJTY) on adjuvant-induced inflammatory arthritis in rats. This issue therefore is a rich resource for all interested in pain and inflammation research and treatment.The researches published in this issue are broad and they accomplished the aim for which the issue was set out. Many manuscripts were received but only those contributing significantly to the subject matter were accepted after thorough peer review. The new insights include topics on systemic models of inflammation contributed by Y. Liu et al., “Effects of Wutou Decoction on DNA Methylation and Histone Modifications in Rats with Collagen-Induced Arthritis,” and X. Wu et al., “Protective Effect of Tetrandrine on Sodium Taurocholate-Induced Severe Acute Pancreatitis.” Z. Shu et al. reported the anti-inflammatory effects of plant extract “Antibacterial and Anti-Inflammatory Activities ofBamidele V. OwoyeleBamidele V. OwoyeleMusa T. YakubuMusa T. YakubuRoi TreisterRoi Treister