**1. Introduction**

Mechanical impact is the leading cause of brain injury, death, and disability in people aged under 45 in the USA, Europe, and developing countries [1]. Road accidents have contributed largely to this aspect. Although bikers are instructed to wear helmets while riding bikes, the quality of helmets still needs to be assessed before their commercialization [2]. Generally, there are three approaches to helmet testing: physical tests, analytical modeling, and numerical simulations [3]. Among them, physical testing has always been accepted worldwide because of its ability to produce real results, incorporating all the given mechanical conditions [4]. Several studies have investigated helmet performances using different impact environments [5]. However, expensive high-speed cameras for impact time measurements and post impact studies were used in these studies.

In countries such as Pakistan, where motorcycles comprise up to 75% of registered vehicles, local helmet manufacturing companies do not have the facilities to test the helmet material's strength and behavior during collision to make their product robust and reliable before bulk production. Existing global testing equipment is expensive and requires specialized expertise. In this study, a pendulum-based impact testing machine for helmets was designed, fabricated, and experimented directly on helmets. Limit switches were used for impact time measurement. The authors found that the results are approaching the standards and are fair to use. Comparing the cost and effectiveness of the machine, this could be a new commercial testing facility for helmets.

**Citation:** Abbas, S.M.; Ali, H.; Naqvi, S.A.A.; Iqbal, M.; Jabbar, H.H.; Azeem, M.; Masud, M. A Novel Low-Cost Machine for Evaluating Helmet Performance in Bike Accident Scenarios. *Eng. Proc.* **2023**, *45*, 39. https://doi.org/10.3390/ engproc2023045039

Academic Editors: Mohammad Javed Hyder, Muhammad Mahabat Khan and Muhammad Irfan

Published: 13 September 2023

#### **2. Materials and Methods**

Table 1 shows the components and their respective materials. The height, length, and width of the machine were assumed to be 244 cm, 180 cm, and 120 cm, respectively.

**Table 1.** Components used in machine and the respective materials.

#### *2.1. Design of Column*

The different machine components were designed using the standard procedures of mechanical design. Columns were designed using the Euler formula given in Equation (1).

$$P\_E = \frac{\pi^2 EI}{L\_e^2} \tag{1}$$

where *PE* is the critical load, *E* is the modulus of elasticity, *Le* is the effective length, and *I* is the moment of inertia. The column was designed with a factor of safety equal to 2. The rectangular column had cross-sections of 30 mm × 60 mm × 2 mm. The height of the column was 244 cm.

#### *2.2. Design of Impactor Arm*

The machine should be as light as possible. Therefore, the impactor arm was made hollow. The safe diameter was calculated using bending criteria with a factor of safety 2. The pin was designed using both shearing and bending criteria where the bending gave a maximum diameter of 10.7 mm. Equation (2) gives the bending criteria.

$$d^3 = \frac{(B.M)(32)}{\pi (S\_y)}\tag{2}$$

The head form was made by filling polyester foam in a head-shaped leather bag over a steel base. The manufactured pendulum-based testing machine is given in Figure 1.

**Figure 1.** (**a**) Head form for helmet rest, (**b**) pendulum-based impact testing machine.

#### *2.3. Impact Duration Measurement*

The duration of the impact was measured using the limit switch along with an Arduino controller. When the contact between the impactor and helmet was established, the current started flowing in the circuit, and it broke the circuit when the two were disconnected. The time span was calculated using the Arduino controller.

#### *2.4. Measurement of Energy Absorbed by Helmet*

The total energy of the impactor upon impact was converted into three energies, i.e., the energy absorbed by the helmet, the post-impact kinetic energy of the helmet, and the remaining energy in the impactor. Equation (3) is used for the calculation of the total energy absorbed in the helmet,

$$E\_T = E\_h + \mathcal{K} \cdot E\_h + E\_{rc} \tag{3}$$

where *ET* is the total energy, *Eh* is the energy absorbed by the specimen, *K*·*Eh* is the kinetic energy of the helmet after impact, and *Erec* is the recoil/remaining energy of the impactor after the collision. The total energy of the impactor was calculated through the maximum kinetic energy achieved by the impactor just before impact. The potential energy of the impactor depended on the height achieved by the impactor measured by measuring the angle of inclination on the protractor attached. The kinetic energy of the helmet was measured through the total distance covered by the helmet on a steel rail, measured using a ruler. The frictional force was responsible for stopping the helmet. The higher the kinetic energy, the higher the distance traveled, as the friction force was assumed to be constant. The following Equations (4) and (5) were utilized to calculate the kinetic energy of the helmet.

$$K \cdot E\_h = -F\_f d\tag{4}$$

$$F\_f = \mu\_k \mathbf{N} \tag{5}$$

#### **3. Results and Discussion**

The impact testing was performed on a standard available bike helmet, along with its catalogue in the local market. The experiments were conducted with varying angles of lift of the impactor for a constant mass of the impactor being taken as 10 kg. Table 2 gives the readings taken from the experiment.

**Table 2.** Experimental results obtained from the pendulum-based impact energy machine.

The results highlight the relationship between the angle of lift of the impactor, the distance traveled by the helmet, and the energy absorbed by the helmet. The potential energy was directly proportional to the angle of lift of the impactor, thereby having a direct relation to both the distance traveled by the helmet and the energy absorbed by it. The apparatus in this study measured the total energy transferred to the helmet, along with the energy transferred to the neck. The limitations, however, include no measurement being taken of the peak acceleration values from the helmet to neck.

The maximum energy absorbed by the helmet was reported to be 110.32 J when the impactor was lifted at a 75◦ angle. The energy absorbed was 80.9 J for a corresponding impact energy of 101.13 J, which was ~80% of the impact energy, including the loss of energy in gaining momentum in the direction of the impact. The energy absorbed was ~88%, as per that given by Bhudolia et al. in 2021, not including the energy transferred to the neck [6].

#### **4. Conclusions**

In this research, a simple method for measuring the impact duration and post-impact energy absorption during helmet impact was presented. A pendulum-based impact energy machine was designed and fabricated, and then the experimentation was conducted with a varying angle of lift of the impactor for a constant mass of the impactor taken as 10 kg. The results concluded that a higher potential energy led to an increase in both the distance traveled by the helmet and the energy absorbed by it. The experimental results were then compared to the reference results and based on this comparison, it can be concluded that the novel machine used in the current study was economical and could be utilized for assessing helmet performance and enhancing the scrutiny of helmet designs.

**Author Contributions:** Conceptualization, S.M.A., H.A., M.A. and M.M.; methodology, S.M.A., H.A., S.A.A.N., M.I. and M.M.; software, S.M.A. and S.A.A.N.; validation, S.M.A. and H.H.J.; formal analysis, S.M.A., H.A., M.A. and M.M.; investigation, S.M.A., H.A., M.A. and H.H.J.; data curation, S.M.A., H.A., M.A. and M.M.; writing—original draft preparation, S.M.A., H.A., S.A.A.N., M.I. and H.H.J.; writing—review and editing, H.A., M.A. and M.M.; supervision, H.A.; project administration, H.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


Control of target specificity by transcriptional repression is also seen in presynaptic photoreceptor cells in the *Drosophila* visual system (Morey et al., 2008). As described above, Caps is expressed in R8 photoreceptor cells and is required for their proper projection to the target layer. Caps expression in R8 cells is positively regulated by the transcription factor Senseless (Sens). In R7 cells, on the other hand, another transcription factor NF-YC represses expression of Sens and the downstream Caps. In NF-YC mutants, R7 cells misexpress Sens and Caps and inappropriately terminate in the R8 target layer as seen when Caps is ectopically expressed in these cells. Thus, repression of Sens and Caps is critical for R7 targeting.

In *tey* mutants, muscle insertion sites of M12 are also specifically affected. Thus, *tey* appears to regulate two key aspects of muscle specification: geometry and synaptic specificity. Several transcription factors have been identified that are expressed in subsets of muscle and/or their progenitor cells (muscle founder cells) and specify the identities of the muscles (reviewed in Tixier et al., 2010). It is an interesting possibility that these genes, like *tey*, may also regulate neuromuscular target specificity. A BTB zincfinger transcription factor, Abrupt (Ab), on the other hand, is expressed in all muscles but regulates specific aspects of motor neuron projection (Hu et al., 1995). In abrupt mutants, ISNb motor neurons reach the target region normally but fail to form appropriate innervation on the target muscles. The differentiation of the target ventral muscles is normal. Ab may thus regulate the expression of general target recognition molecules, such as Haf, in these muscles. Several transcription factors that function in motor neurons to specify the target domain have also been identified, including Even-skipped, Islet and Lim3 (reviewed in Landgraf and Thor, 2006). However, whether these transcription factors regulate the expression of target recognition molecules remain unknown.

## **PARTNER RECOGNITION BY MYOPODIA**

In addition to recognition by presynaptic motor neurons, recent studies suggest that the postsynaptic muscles also seek and find their presynaptic partners. In fixed specimens, differentiated muscles show little sign of motility and, therefore, have long been regarded as passive players during synaptic matchmaking, waiting

for the arrival of motor neurons. However, Ritzenthaler et al. (2000) used high-resolution live imaging to show that muscles actually bear a number of dynamic protrusions, called myopodia, that extend and retract prior to and during the period of neuromuscular interaction. Myopodia are actin-based membrane microprocesses ∼10µm or longer in length and 0.1–0.2µm in diameter. Myopodia initially extend in random directions but gradually cluster at the site of innervation as they interact with the presynaptic filopodia (**Figure 4A**, top). The clustering of myopodia depends on interaction with the presynaptic motor neurons (Ritzenthaler et al., 2000; Ritzenthaler and Chiba, 2003).

As myopodia cluster, they intermingle and "zip" with the presynaptic filopodia and finally fuse to form the nascent postsynaptic sites (Ritzenthaler et al., 2000; Ritzenthaler and Chiba, 2003; Kohsaka et al., 2007). Thus, neuromuscular targeting is a reciprocal process in which motor neurons and muscles seek each other.

What is the precise role of myopodia? Cells use filopodia and other cellular extensions to efficiently transmit and receive information via long-distance direct cell–cell interaction (Rørth, 2003; Hsiung et al., 2005; Heiman and Shaham, 2010; Roy et al., 2011). For example, neuronal growth cones extend filopodia as they explore the local environment. In the *Drosophila* imaginal disc, cytonemes and other actin-based extensions are utilized not only in the signal-receiving cells to actively seek cues from a distance but also in the signal-sending cells to efficiently present information to the receiving cells (Rørth, 2003; Hsiung et al., 2005; Roy et al., 2011). Similarly, myopodia may function to receive and/or send information at a distance. A study by Kohsaka and Nose (2009) showed that myopodia have a sensory function. Simultaneous live-imaging of muscles and motor axons is used to study the behavior of myopodia when they encounter partner or non-partner motor neurons (**Figure 4B**). When myopodia encounter the growth cones of partner motor neurons, about half of the contacts are stabilized to from synapses. In contrast, none of the contacts with the non-partners are stabilized, suggesting that individual myopodia can recognize the appropriate partner. So, just like presynaptic growth cones seek and find the right targets, postsynaptic muscles also select among potential presynaptic partners using myopodia.

Localization and function of Caps in myopodia has also been studied (Kohsaka and Nose, 2009). Caps is localized at the tip of myopodia, where many of the initial contacts between muscles and growth cones take place (**Figure 4C**). In *caps* mutants, fewer myopodial contacts with the presynaptic motor neurons are stabilized in M12. The size of the nascent presynaptic terminal is also reduced. Similar but more penetrant phenotypes are seen in *caps, trn* double mutants (**Figure 4A**, bottom). Thus, Caps-mediated signaling at the tip of myopodia appears to be critical for selective synapse formation. It is likely that Caps functions in a bi-directional manner at the tips of myopodia (**Figure 4B**, right). By localizing at the tip of myopodia, Caps may be efficiently presented to and recognized by the growth cones. Caps may also be involved in the recognition of motor neurons by the myopodia. Such bidirectional signaling might ensure proper synaptic matchmaking. Many signaling and adhesion molecules are known to accumulate at the tips of filopodia, including Ena/VASP, formins, and integrins (Faix and Rottner, 2006; Mattila and Lappalainen, 2008). Thus, tips of myopodia may function as a signaling center that regulate myopodia dynamics and synapse formation. Dendrites of vertebrate neurons also cast out dendritic filopodia during synapse formation. A recent study in rodents showed that dendritic filopodia of hippocampal neurons discriminate between target and non-target axons (Lohmann and Bonhoeffer, 2008). Regulation of synapse specificity by postsynaptic protrusions thus seems to be a common mechanism across taxa.

## **CONCLUSION**

When investigators began searching for NMJ target recognition molecules in the 1990s, most envisioned a simple lock-and-key mechanism, as initially postulated by Sperry (1963). However, the dance between motor neurons and target muscles turns out to be far more complex than initially thought. Multiple molecular chaperons appear to mediate the partner selection in a partially redundant and combinatorial manner. Partner selection is not only determined by attraction to the partner but also by exclusion from other cells. Furthermore, the molecular courtship is not one-sided but is a mutual searching process mediated by complex interactions of filopodia and myopodia.

Studies thus far implicate at least 11 proteins in targeting of M12, including the attractive cues Caps and Tartan on the muscle, inhibitory cues Wnt4, Toll, Beat-3c, Glt, Lsp2, Sulf1, and CG6867 on the neighboring M13, and general cues Haf and CG8561 on all muscles. Why so many target cues are present to specify the targeting of a single muscle is a mystery. Determining how multiple classes of molecular cue are translated into a coherent downstream signal that culminates in the formation of specific synapses is an important future direction. Development of a complete set of GOF constructs for all cell surface and secreted proteins would allow the definition of every protein that can produce a neuromuscular targeting GOF phenotype and provide a list of candidate players. Even more mysterious is how the expression of diverse target cues is coordinated during muscle development. Although studies of Tey begin to answer this question, much more work is needed to obtain the whole picture. Clues may be obtained by comprehensive analyses of the transcriptional cascade, like those performed in the *Drosophila* mesoderm (Zinzen et al., 2009; Junion et al., 2012).

## **NOTE ADDED IN REVISION**

Mosca et al. (2012) recently reported that Teneurin (Ten)-m and Ten-a, belonging to conserved epidermal growth factor (EGF) repeat-containing transmembrane proteins, regulate neuromuscular targeting in *Drosophila*. Ten-m and Ten-a are expressed in all muscles and motor neurons, respectively, and mediate bidirectional interaction required for proper organization of the NMJs. Ten-m is expressed at a higher level in muscle 3 and 8, and are required for synapse formation in these muscles. Like Fas2, changing the relative level of Ten-m between muscles 6 and 7 alters the pattern of target selection. Thus, Ten-m and Ten-a appear to regulate general synaptic organization as well as target specificity.

## **ACKNOWLEDGMENTS**

I thank present and past members of the lab for their contribution to the studies described in this article and Kasumi Shibahara for the help in illustration. This article is supported by a Grantin-Aid for Scientific Research on Innovative Areas "Mesoscopic Neurocircuitry" (No. 22115002) and for Scientific Research on Innovative Areas "Comprehensive Brain Science Network" (No. 221S0003), of The Ministry of Education, Culture, Sports, Science, and Technology, Japan, and for Scientific Research (B) (No. 23300114) of Japan Society for the Promotion of Science (JSPS), to Akinao Nose.

## **REFERENCES**

leucine-rich repeat proteins in the development and function of neural circuits. *Annu. Rev. Cell Dev. Biol.* 27, 697–729.

wing imaginal disc cytonemes on Decapentaplegic. *Nature* 437, 560–563.

A neural tetraspanin, encoded by late bloomer, that facilitates synapse formation. *Science* 271, 1867–1870.

S. L. (2008). Coordinate control of synaptic-layer specificity and rhodopsins in photoreceptor neurons. *Nature* 456, 795–799.

recognition. *Int. Rev. Neurobiol.* 75, 1–31.

(2002). Forked end: a novel transmembrane protein involved in neuromuscular specificity in *Drosophila* identified by gain-offunction screening. *J. Neurobiol.* 51, 205–214.

predicts spatio-temporal cisregulatory activity. *Nature* 462, 65–70.

**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 08 March 2012; paper pending published: 28 March 2012; accepted:* *23 April 2012; published online: 08 May 2012.*

*Citation: Nose A (2012) Generation of neuromuscular specificity in Drosophila: novel mechanisms revealed by new technologies. Front. Mol. Neurosci. 5:62. doi: 10.3389/fnmol.2012.00062*

*Copyright © 2012 Nose. This is an open-access article distributed under the terms of the Creative Commons Attribution Non Commercial License, which permits non-commercial use, distribution, and reproduction in other forums, provided the original authors and source are credited.*

## Transgenic strategy for identifying synaptic connections in mice by fluorescence complementation (GRASP)

## *Masahito Yamagata and Joshua R. Sanes\**

*Department of Molecular and Cellular Biology and Center for Brain Science, Harvard University, Cambridge, MA, USA*

#### *Edited by:*

*Joshua A. Weiner, The University of Iowa, USA*

#### *Reviewed by:*

*Christian Lohmann, Netherlands Institute for Neuroscience, Netherlands James Jontes, Ohio State University, USA*

#### *\*Correspondence:*

*Joshua R. Sanes, Department of Molecular and Cellular Biology and Center for Brain Science, Harvard University, 52 Oxford Street, Cambridge, MA 02138, USA. e-mail<EMAIL_ADDRESS>
In the "GFP reconstitution across synaptic partners" (GRASP) method, non-fluorescent fragments of GFP are expressed in two different neurons; the fragments self-assemble at synapses between the two to form a fluorophore. GRASP has proven useful for light microscopic identification of synapses in two invertebrate species, *Caenorhabditis elegans* and *Drosophila melanogaster*, but has not yet been applied to vertebrates. Here, we describe GRASP constructs that function in mammalian cells and implement a transgenic strategy in which a Cre-dependent gene switch leads to expression of the two fragments in mutually exclusive neuronal subsets in mice. Using a transgenic line that expresses Cre selectively in rod photoreceptors, we demonstrate labeling of synapses in the outer plexiform layer of the retina. Labeling is specific, in that synapses made by rods remain labeled for at least 6 months whereas nearby synapses made by intercalated cone photoreceptors on many of the same interneurons remain unlabeled. We also generated antisera that label reconstituted GFP but neither fragment in order to amplify the GRASP signal and thereby increase the sensitivity of the method.

**Keywords: GRASP, GFP, synapse, retina, photoreceptor, rod, neuroligin, neurexin**

## **INTRODUCTION**

Analysis of neural circuits requires identification of neurons that are synaptically connected to each other. A major impediment to mapping these connections is that many neurites in the central nervous system are too small to be resolved by conventional light microscopy: synaptic sites can be visualized but the neurites from which they arise cannot be identified unambiguously. At present, therefore, connectivity is generally assessed by electron microscopic and electrophysiological methods. Both of these techniques are extremely laborious, however, and both are difficult to apply when the pre- and postsynaptic somata are separated by long-distances. Accordingly, many groups have sought improved light microscopic methods for circuit analysis, and three have shown considerable promise. The first is super-resolution microscopy, sometimes called nanoscopy, in which even the most slender neurites can be resolved (Huang et al., 2010). The second is transsynaptic tracing, in which genetically encoded labels are secreted at synaptic sites and selectively internalized by the apposed pre- or postsynaptic membrane (Horowitz et al., 1999; Yoshihara et al., 1999; Wickersham et al., 2007; Lo and Anderson, 2011). The third is "GFP Reconstitution Across Synaptic Partners" or GRASP (Feinberg et al., 2008).

GRASP is based on the use of non-fluorescent fragments of GFP that can be fused to other proteins, yet retain the ability to self-assemble and form a fluorophore. This complementation strategy was initially used to detect intracellular protein-protein interactions (Hu and Kerppola, 2003; Zhang et al., 2004; Cabantous et al., 2005). To adapt the method for detection of synapses, Feinberg et al. (2008) used a GFP derivative that had been engineered for efficient complementation ("superfolder GFP"; Pedelacq et al., 2006), split it into two fragments (sGFP1–10 and sGFP11), fused them to the ectodomains of cell surface proteins, and expressed one fusion protein in each of two neurons in *C. elegans* that were known to be synaptically connected. They showed that the fragments reconstituted fluorescent GFP specifically at sites of synapses between the two neurons. Subsequently, Gordon and Scott (2009) and Gong et al. (2010) used the method to mark synapses in *Drosophila*.

GRASP is particularly useful because it is the only light microscopic method to date capable of unequivocally marking synaptic contacts between two genetically specified neurons. We, therefore, asked whether it can be applied to mammals. We report here generation of knock-in mice in which sGFP1–10 and sGFP11 are fused to components of synaptic membrane proteins, neuroligin (NLG), and neurexin (NXN) (Südhof, 2008), and expressed in mutually exclusive neuronal subsets. We demonstrate that GRASP specifically marks synapses between members of the two subsets in retina, without marking synapses made by other axons on the same postsynaptic cells.

## **MATERIALS AND METHODS GRASP CONSTRUCTS**

We modified the DNA sequence of superfolder GFP (Pedelacq et al., 2006) by optimizing codon usage for mice and removing potential splicing donor and acceptor motifs without altering the encoded amino acid sequence (GenBank accession number JQ341914). The modified gene was synthesized (DNA2.0, Menlo Park, CA) and cloned into a backbone derived from CMV-EGFP-N1 (Clontech, Mountain View, CA). The GFP1–10 and GFP11 fragments were retrieved from this vector by PCR and fused to rat neuroligin-1 or neurexin-1β (obtained from Alice Ting, MIT), or human CD4 (obtained from Evan Feinberg and Cori Bargmann, Rockefeller University).

## **CELL LINES**

Human embryonic kidney 293 cells (HEK293; ATCC, Manassas, VA) were subcloned and a derivative was selected that could be cloned with high efficiency; we call this line 293PL. 293PL cells in 24-well tissue culture dishes were transfected using DMRIE-C (Life Technologies, Grand Island, NY), then trypsinized 3 days later, plated on 10 cm tissue culture dishes, and selected in 1 mg/ml G418 (Life Technologies). Colonies were split and tested by immunostaining for expression of the gene product. More than 70% of the cells in each clone showed high expression of the introduced gene.

Each clone was cultured to confluency, trypsinized, and plated onto glass coverslips, either alone or mixed with cells of a second clone. After 2–3 days, cultures were fixed with 100% methanol or 4% paraformaldehyde/PBS for 15 min at room temperature, then rinsed with PBS. Methanol-fixed cultures had lower background than paraformaldehyde-fixed cultures, so this treatment was usually used for detecting native GRASP signals. For immunostaining, paraformaldehyde cultures were processed as described previously (Yamagata and Sanes, 2010). Finally, cultures were mounted with FluoroGel (Electron Microscopy Sciences, Hatfield, PA) and imaged with a Pascal LSM-510 confocal microscope (Zeiss).

Use of stably transfected lines was required for these assays because co-expression of two complementary split GFP fragments in the same cell leads to efficient *cis*-reconstitution that hampers detection of the much lower levels of *trans*reconstitution between apposed cells. Surprisingly, we observed such co-expression when we mixed populations that had been transiently transfected with complementary fragments. We suspect that low levels of DNA adhere to cells even when the cells were trypsinized and rinsed multiple times prior to co-culture.

## **ANTIBODIES TO GFP**

Full length superfolder GFP was cloned into pEcoli-6xHN (Clontech) and introduced into BL21(*DE3*) pLysS cells. The cells were grown in Magic Media (Life Technologies) and processed with Bugbuster (EMD Chemicals, Philadelphia, PA), and the GFP was purified with Talon columns (Clontech). Hens were immunized with the recombinant GFP (Covance), eggs from immunized hens were harvested, and the IgY fraction from egg yolk was purified with IgY purification kit (Pierce, Rockford, IL).

To obtain antibody that reacted selectively with holo-GFP, the sGFP1–10 fragment was expressed in *E. coli* using pEcoli-6xHN. This fragment was insoluble, so inclusion bodies were purified, dissolved with 4M urea (in 50 mM TrisHCl, pH 7.4), purified using Talon columns, and coupled to NHS-Activated Agarose (Pierce). The anti-GFP IgY was absorbed to this resin and the non-binding fraction was further absorbed with acetone powder prepared from mouse brain. This purified antibody is called anti-rGFP.

A hybridoma producing a mouse monoclonal antibody GFP-G1 (subclass, IgG1) was obtained by immunizing a female Balb/c mouse. Its splenocytes were fused to FOX-NY myeloma line, and selected by a standard procedure for generating hybridomas. Hybridomas were screened by staining tissue from GFP-expressing mice to obtain an antibody suitable for immunostaining. One antibody selected, GFP-G1, recognizes all the GFP derivatives tested (EYFP, Venus, superfolder GFP, and cerulean). IgG was purified from cultured supernatants using Protein-G affinity gels (GenScript, Piscataway, NJ).

## **KNOCK-IN MOUSE LINE**

To generate a GRASP knock-in targeting vector, pRosa26-PAS (Srinivas et al., 2001) was modified in the following series of steps. (1) A phosphorylated i-SceI linker (AGTTACGCTAGGGATAA CAGGGTAATATAG) was ligated into the SwaI site, producing a recognition site for linearization of the targeting vector. (2) A PacI-FRT-neo-FRT-AscI selection cassette was cloned into pROSA26PAS-i-SceI. (3) A PacI-CAG-RfA-WPRE-PacI cassette containing the chicken β-actin promoter and CMV immediate-early enhancer (together called CAG), a Gateway RfA destination cassette (Gateway Vector Conversion system; Life Technologies), and a WPRE fragment (woodchuck hepatitis virus post-transcriptional element) was assembled in a modified pBluescriptKS+ that had been generated by inserting GA GCTCAGTTACGCTAGGGATAACAGGGTAATATAGAATTCTT AATTAAGCGGCCGCGATCGCCCGGGCATTTAAATGGCCTG CAGGGCCGTTTAAACGGCCGGCCGTCGACTCGAGCGTAA CTATAACGGTCCTAAGGTAGCGAAGGTACC into the Kpn I/SacI sites. (4) This cassette was cloned into a PacI site of pROSA26PAS-FNF-iSceI. (5) sGFP11::NXN was cloned to pCR8- Topo (Life Technologies) with an appended I-CeuI sequence at its *N*-terminus. (6) sGFP1–10::NLG and a polyadenylation signal were cloned into a modified pBluescript+ that had generated by inserting CCGGGAGCTCCGTAACTATAACGGTCCTAAGGTA GCGAATTCTTAATTAAGCGGCCGCGATCGCCCGGGCATTT AAATGGCCTGCAGGGCCGTTTAAACGGCCGGCCGTCGAC TCGAGCGTAACTATAACGGTCCTAAGGT AGCGAAGGTACC GCGC and 2 loxP sites into the KpnI/SacI sites. (7) This sGFP1– 10::NLG with a polyadenylation signal was excised with I-CeuI and cloned into an I-CeuI site of 11::NXN in pCR8-Topo. (8) This sGFP-10::NLG-sGFP11-NXN cassette was transferred to the product of step 4 above using LR clonase (Life Technologies).

The i-SceI linearized vector was electroporated into a 129/B6 F1 hybrid ES cell line, V6.5. G418-resistant, targeted ES clones were identified by PCR. Correct targeting efficiency was *>*50%. ES cell transfections and blastocyst injections were performed by the Genome Modification Facility, Harvard University. After germ-line transmission, the FRT-neo-FRT sequence was removed by crossing to mice that express Flp recombinase ubiquitously (Farley et al., 2000).

Rhodopsin-Cre transgenic mice (Li et al., 2005) were obtained from Shiming Chen (Washington University). Animal procedures were in compliance with the US National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Animal and Care and Use Program at Harvard University.

#### **IMMUNOSTAINING**

Immunostaining was performed as described by Yamagata and Sanes (2010). Briefly, tissues were dissected, fixed in 4% paraformaldehyde/PBS overnight at 4◦C, sunk in 15% (w/v) and 30% (w/v) sucrose/PBS, mounted in OCT compound, and cryosectioned. The sections were treated with 0.1% Triton X-100/PBS followed by Image-iT FX signal enhancer (Life Technologies), blocked with 5% skim milk/PBS, incubated with primary antibodies overnight at 4◦C, rinsed, incubated with secondary antibodies with Neurotrace 435 (Life Technologies) overnight at 4◦C, rinsed, and mounted with FluoroGel.

Primary antibodies used were: rabbit anti-Cre (Abcam, Cambridge, MA); mouse anti-neuroligin-1 and anti-neurexin-1β (NeuroMab, Davis, CA); mouse anti-bassoon (GeneTex, Irvine, CA); mouse anti-SV2 (Developmental Studies Hybridoma Bank, Iowa City, IA), mouse anti-β-dystroglycan (Leica Microsystems, Buffalo Grove, IL), mouse anti-PSD95 (Affinity Bioreagents, IgG1); monoclonal anti-GFP (GFP-Mab20, Sigma) and the two anti-GFP antibodies described above. Rhodamineconjugated Peanut agglutinin was from Vector Lab (Burlingame, CA) and secondaryantibodieswerefrom Jackson Immunoresearch Laboratories (West Grove, PA).

## **RESULTS**

### **GRASP BETWEEN CULTURED MAMMALIAN CELLS**

Superfolder GFP (Pedelacq et al., 2006) was designed for use in *E. coli*. To improve expression in mammalian cells, we optimized its codon usage for mice (GenBank, JQ341914), and fused the sGFP1–10 and sGFP11 fragments to mammalian transmembrane cell surface proteins: human CD4 glycoprotein with a cytoplasmically fused monomeric cherry, rat neurexin-1β (NXN) and rat neuroligin-1 (NLG; **Figure 1A**). The CD4-cherry fusion has been used for GRASP in *C. elegans* (Feinberg et al., 2008) and *Drosophila* (Gordon and Scott, 2009), and NXN and NLG are localized to the pre- and postsynaptic membranes, respectively, of most mammalian synapses (Südhof, 2008; Shen and Scheiffele, 2010).

The six fusions were introduced into HEK293PL cells and stably transfected populations were selected, passaged and cultured either alone or in pairwise combinations. We observed no reconstituted GFP fluorescence when any one of the six populations was cultured alone or in most combinations (**Figure 1** and **Table 1**). In only two cases were contacts between cells fluorescent, indicating reconstitution of GFP in mixtures of sGFP1–10::NLG cells with sGFP11::NXN cells and in mixtures of sGFP1–10::NXN cells with sGFP11::NLG cells (**Figure 1D** and **Table 1**). GRASP signal was readily detectable in live cells viewed with an inverted microscope, supporting the possibility that the technique could be used to image synapses in live tissue. Immunolabeling with antibodies to NXN and NLN confirmed that in each case, signal was present only at contacts between

## **FIGURE 1 | GRASP signals in cultured mammalian cells.**

**(A)** Superfolder GFP fragments sGFP1–10 and sGFP11 were fused to mammalian transmembrane proteins neurexin (NXN), neuroligin (NLG), and CD4 for display on the cell surface. Assembly of sGFP1–10 with sGFP11 reconstitutes fluorescent GFP. **(B–D)** 293 cells stably transfected with either

1–10::NLG **(B)** or 11::NXN **(C)** show no GRASP fluorescence. However, GRASP signal was observed at contacts between sGFP1–10::NLG- and sGFP11::NXN-expressing cells **(D)**. **(D')** shows the outline of cells in **(D)**. **(E–G)** No complementation was detected with CD4 fusions. Bar: 10μm.

**Table 1 | GRASP signal at cell-cell contacts of various configurations.**

*Stably transfected 293 cell lines were co-cultured as indicated.*

+*, GRASP signal was observed;* −*, GRASP signal was not observed.*

NXN-expressing and NLN-expressing cells (data not shown). The failure to observe complementation between sGFP1–10::CD4 and sGFP11::CD4, was surprising in that these fusions generate GRASP signals in *C. elegans* and *Drosophila* (Feinberg et al., 2008; Gordon and Scott, 2009).

### **GENERATION OF GRASP KNOCK-IN MICE**

To mark synapses *in vivo*, we generated mice in which sGFP1– 10::NLG and sGFP11::NXN are expressed in complementary sets of neurons. Tests in HEK293PL cells had shown that the two fragments complement efficiently when expressed in the same cell, resulting in intense signals that prevent detection of the relatively weak signals resulting from complementation at opposed membranes between cells (see Methods). To prevent such co-expression, we designed a vector that contained both fragments, with sGFP1–10::NLG expressed by default and sGFP11-NXN expressed only following Cre-dependent excision of sGFP1–10::NLG (**Figures 2A,B**). The cassette (CAG-LoxPsGFP1–10::NLG-pA-LoxP-11:: sGFP11::NXN-pA) was introduced into HEK293PL cells using a piggyBac-transposon (Yamagata and Sanes, 2008, 2010) to generate a cell line harboring a single copy. The cells expressed sGFP1–10::NLG but not sGFP11::NXN. Following transfection with a plasmid encoding Cre, the cells expressed sGFP11::NXN but not sGFP1–10::NLG (data not shown).

We then generated mice using this gene switch vector. We used a knock-in strategy to ensure that the genome contained only a single copy of the cassette. Thus, any individual cell can express either sGFP1–10::NLG or sGFP11::NXN, but can never express both. In contrast, transgenes introduced by oocyte injection often comprise multiple copies in tandem, so recombination of only a subset of the copies would lead to *cis*-complementation. To maximize levels of expression, we used the strong composite CAG (CMV + chicken β-actin) promoter-enhancer, added a WPRE to stabilize mRNA, and inserted the vector into the ROSA26 locus, which is suitable for ubiquitous expression of transgenes (Soriano, 1999; Zong et al., 2005; Madisen et al., 2010). Homologous recombinants were selected and used to generate germ-line chimeras by standard methods. We call the resulting line mGRASP1.

For initial tests, we used a transgenic line that expresses Cre recombinase specifically in rod photoreceptors under control of regulatory elements from the rhodopsin gene (Li et al., 2005). Immunostaining for Cre recombinase confirmed the specificity of the transgene (**Figures 2D,H**). We expected that sGFP1–10::NLG would be broadly expressed and sGFP11-::NXN would be undetectable in mGRASP1 mice, whereas sGFP11- NXN would be expressed only in rods, and sGFP1–10::NLG would be expressed in all other retinal cells in double transgenic mGRASP1; rhodopsin-Cre mice (**Figure 2C**). Indeed, in mGRASP1 mice, NLG immunoreactivity was present throughout the retina, including in photoreceptor outer segments (**Figure 2E**). NLG immunoreactivity was barely detectable in wild-type retina (data not shown), indicating that the signal in mGRASP1 mice reflected expression of sGFP1–10::NLG. Little NXN reactivity was detected in retinas of wild-type or mGRASP1 mice. In mGRASP1; rhodopsin-Cre mice, NLG immunoreactivity was lost from outer retina, confirming loss of sGFP1– 10::NLG (**Figures 2E,I**). Likewise, immunostaining with NXN confirmed accumulation of sGFP11::NXN in the outer plexiform layer of mGRASP1; rhodopsin-Cre mice (**Figures 2L,M**). Most important, complementation led to appearance of GRASP signal in the outer plexiform layer of mGRASP1 (**Figures 2F,G,J,K**); rhodopsin-Cre mice; this is layer in which axon terminals of photoreceptors form synapses on dendrites of bipolar and horizontal cells (Rao-Mirotznik et al., 1995; Sterling and Matthews, 2005).

## **IMMUNOAMPLIFICATION OF GRASP SIGNALS**

Although GRASP signals in mGRASP1; rhodopsin-Cre mice were clear and specific, they were weak. We attempted to enhance the signal using a commercially available monoclonal antibody, GFP-Mab20, which binds to an epitope present in holo-GFP but not in either the sGFP1–10 or the sGFP11 fragment (Gordon and Scott, 2009). This antibody was useful but inadequate, because it recognizes only a single epitope and therefore, provides limited amplification, and also because anti-mouse secondary antibodies lead to non-specific staining of mouse tissues. We, therefore, generated polyclonal antisera to native GFP, and purified from the sera those antibodies that recognized native GFP but not sGFP1– 10 (see Methods and **Figures 3A–D**). The purified antibodies specifically recognized reconstituted GFP at borders between cells transfected with sGFP1–10::NLG and sGFP11::NXN cells, whereas a monoclonal antibody to GFP, which we also generated, recognized both the reconstituted GFP and the sGFP1–10::NLG fragment (**Figures 3E–G**). We used the holo-GFP-specific antibody, which we call anti-rGFP, for further characterization of mGRASP1 mice.

**FIGURE 2 | Generation of GRASP knock-in mice. (A)** Targeting construct for generation of mGRASP1 mice. sGFP1–10::NLG was placed between LoxP sites, followed by sGFP11::NXN. WPRE, Woodchuck post-transcriptional regulatory element; pA, polyadenylation signal. FRT-SV40-neo-FRT and DTX denote selection cassettes used for positive and negative selection of correctly targeted ES cells, respectively; these cassettes were absent from the final mGRASP1 line. **(B)** Cre recombinase deletes sGFP1–10::NLG, thereby activating expression of sGFP11::NXN. **(C)** Diagram of gene switching in retina. sGFP1–10::NLG is expressed in all cells of mGRASP1 mice (top). Following cross to rhodopisin-Cre, mice, expression of sGFP1–10::NLG is extinguished in rod photoreceptors but persists in other cells; concurrently, expression of sGFP11::NXN is activated specifically in rod photoreceptors. **(D–G)** Sections of retina from P30 mGRASP mice. **(D,E)** Sections stained with anti-Cre **(D)**, and anti-NLG **(E)**. Neurotrace 435 counterstain is blue. No Cre is detectable, but NLG immunoreactivity is present throughout the retina. Because NLG immunoreactivity is barely

detectable in wild-type retina (not shown). signal in **(E)** is due to sGFP1–10::NLG. Note strong signal associated with photoreceptor outer segments (arrowheads in **E**). **(F,G)** No GRASP signals are detectable in retina. **(G)** shows high-power view of OPL from area boxed in **(F)**. **(H–K)** Sections of retina from P30 mGRASP; rhodopsin-Cre mice. **(H,I)** Sections stained with anti-Cre **(H)**, anti-NLG **(I)**, and Neurotrace 435 (blue). Cre is abundant in photoreceptor nuclei in the ONL **(H)** and NLG immunoreactivity is lost from photoreceptor outer segments (arrowheads in **I**). **(J,K)** GRASP signals are detectable in OPL. **(K)** shows high-power view of area boxed in **(J)**. **(L,M)** Sections of retina from P30 mGRASP; rhodopsin-Cre mice were stained with anti-NLG **(L)** and anti-NXN **(M)**. NXN is concentrated in nerve terminals in the OPL. Because NXN immunoreactivity is barely detectable in wild-type retina (not shown), signal in **(M)** is due to sGFP11::NXN. Bar, 10μm for **D,E,H,I**; 5μm for **F,J**; 2.5 μm for **G,K–M**. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.

5μm for **(G)**.

**reconstituted GFP. (A,B)** Superfolder GFP is fluorescent when expressed in 293 cells **(A)** whereas split GFP1–10 is not **(B)**. Chicken antibodies to bacterially expressed superfolder GFP recognize both proteins **(A',B')**. Blue channel shows counter-staining with Neurotrace 435. **(C,D)** Following absorption to sGFP1–10 and tissue, the same antibodies react with fluorescent superfolder GFP **(C,C')**, but not sGFP1–10 **(D,D')**. **(E,F)** Mouse

**CHARACTERIZATION AND SPECIFICITY OF GRASP SIGNALS IN PHOTORECEPTOR SYNAPSES**

To test whether the GRASP signal in the outer plexiform layer of mGRASP1; rhodopsin-Cre mice is localized to rod synapses, we double-labeled sections with anti-rGFP plus antibodies to synaptic components. Bassoon marks active zones in photoreceptor nerve terminals (Brandstätter et al., 1999). GRASP signals abutted Bassoon-positive puncta (**Figures 4A,B**). In one set of sections from P60 retina, we found that 73 ± 4% of Bassoon-positive puncta in the OPL were anti-rGFP-positive. This is an underestimate, because Bassoon marks cone as well as rod synapses and the latter are unlabeled (see below). In many cases, a pair of GRASP-positive puncta were localized directly adjacent to a larger Bassoon-positive punctum. In these triplets, the Bassoon-positive structure was generally closer to the outer nuclear layer, in which photoreceptors reside, while the rGFP-positive puncta were closer to the inner nuclear layer, which contains horizontal and bipolar cells. It is known that each rod synapse comprises a presynaptic rod terminal, invaginating processes from two horizontal cells, and a central bipolar process; the horizontal cell processes directly about the photoreceptor membrane whereas the bipolar process does not (**Figure 4I**; also see Rao-Mirotznik et al., 1995; Sterling and Matthews, 2005). Thus, the pattern we observed suggests that GRASP signals mark direct contacts between the rod terminal and the horizontal processes. Lack of GRASP signal associated with the bipolar process is consistent with the idea that directly abutting

GFP-G1 stained the sGFP1–10::NLG expressing cell. In contrast, anti-rGFP stains only contacts between sGFP1–10::NLG- and sGFP11::NXN-expressing cells (mab GFP-G1-positive and -negative, respectively). Bar, 20μm for **A–F**;

membranes are required for assembly of sGFP1–10 with sGFP11. We also stained sections with antibodies to PSD95 and βdystroglycan. In many central synapses, PSD95 is associated with the postsynaptic membrane, but in photoreceptor synapses it is localized in perisynaptic areas within photoreceptor nerve terminals, adjacent to active zones (Koulen et al., 1998; Yamagata and Sanes, 2010). Likewise, β-dystroglycan is present in expanded portions of photoreceptor terminals, but not at active zones (Blank et al., 1999). Double labeling revealed that rGFP-positive puncta were near PSD95- and β-dystroglycan-positive puncta (**Figures 4C–F**). These patterns support the idea that GRASP signals line rod nerve terminals.

Finally, we asked whether GRASP signals are confined to synapses made by rod photoreceptors onto horizontal cells, or whether they are also present at closely apposed synapses made by cone photoreceptors on the same horizontal cells. In mice, 95–97% of photoreceptors are rods, and only 3–5% are cones, so if GRASP signals were not confined to appropriate synapses on horizontal cells we would expected them to be present in nearby cone synapses. To mark cone pedicle synapses, we incubated sections with peanuts agglutinin (PNA), which labels cone but not rod terminals in mouse retina (Blanks and Johnson, 1983). GRASP signals were excluded from PNA-positive structures (**Figures 4G,H**), indicating that the method can distinguish synapses made by two different presynaptic cells onto the same postsynaptic cell.

#### **LONG-TERM EXPRESSION OF MAMMALIAN GRASP SYSTEM**

The GRASP method generates a transsynaptic link that is not present endogenously. It is, therefore, a concern that the long-term presence of this link could affect synaptic structure. Moreover, our implementation of the GRASP method requires expression of NXN and NLG both of what can affect synapse number when overexpressed (Chubykin et al., 2007; Dahlhaus et al., 2010). We, therefore, analyzed the number and molecular architecture of rod photoreceptor synapses in 7 month-old

mGRASP1; rhodopsin-Cre mice. The presence of GRASP links over this prolonged period had no detectable effect on the levels or localization of any synaptic marker tested, including SV2, bassoon, PSD95, and β-dystroglycan (**Figure 5**). In addition, the number of photoreceptor terminals (bassoon-positive puncta) in the OPL of 7 month-old retinas did not differ significantly between mice expressing both mGRASP and Rhodopsin-Cre, and controls expressing only Rhodopsin-Cre (8*.*9 ± 2*.*3 and 9*.*1 ± 2 puncta/100 μm2, respectively; *p >* 0*.*1 by *t*-test). Thus, long-term expression of GRASP linkages at synapses has no detectable effect on the persistence or molecular architecture of these synapses.

### **DISCUSSION**

We have shown that the GRASP method, which was originally developed to detect synaptic connections in *C. elegans* (Feinberg et al., 2008), can also be used to detect synapses in mice. As a test, we labeled synapses between rod photoreceptors and their postsynaptic partners, horizontal cells; we showed that labeling is specific in that synapses made by cone receptors on the same postsynaptic partners were unlabeled.

While we were preparing this work for publication, Kim et al. (2012) described an alternative method for implement GRASP in mice. They, like us, found that fusing fragments to NXN and NLG was effective whereas fusions to CD4 were ineffective.

In our cases, GRASP signals from CD4 fusions were unworkable faint, whereas for Kim et al. (2012), signals were detectable but not synaptically localized. Whereas we used a transgenic strategy to express the split GFP fragments, they used a viral strategy. A major advantage of the transgenic strategy is that labeling is completely non-invasive whereas a major advantage of the viral strategy is that post- as well as presynaptic cells can be selected. Another difference between the methods is that whereas we used unmodified NXN and NLG, they mutated NLG to prevent strong interactions with NXN, which, as noted above, might affect synaptic size or strength (Chubykin et al., 2007; Dahlhaus et al., 2010). Although we have noted no ill effects of overexpression, it may be more prudent to adopt the strategy of Kim et al. (2012) in the future.

Together the two sets of results make a strong case that GRASP will be generally useful for circuit analysis in mice. First, both transgenic (this paper) and viral methods (Kim et al., 2012) can be used to deliver GRASP components. Second, both light (this paper) and electron microscopic (Kim et al., 2012) methods demonstrate the specificity of GRASP labeling. Third, marking of synapses in retina (this paper) and forebrain (Kim et al., 2012) indicate that the method can be applied to multiple synaptic types.

The major drawback to our instantiation of the GRASP method is that it is insufficiently sensitive. Although signals in photoreceptor synapses are robust, these are especially large synapses, and we have not been able to consistently detect signals at smaller synapses in the inner plexiform layer of the retina, the spinal or the cortex using other Cre transgenic lines. We suspect that this limitation reflects the relatively low expression of the postsynaptic partner, sGFP1–10::NLG in mGRASP1 mice. This, in turn, may result from fact that the WPRE in the construct, which greatly enhances expression (Madisen et al., 2010) would be expected to stabilize the mRNA encoding sGFP11::NXN but not that encoding sGFP1–10::NLG. In addition, our experience with gene transfer methods *in vivo* suggests that the viral and electroporation methods used by Kim et al. (2012) lead to considerably higher levels of expression than does the knock-in method we used. To circumvent this limitation, and thereby expand the range of applications for the method, we are currently engineering transgenes in which both components are expressed at higher levels.

### **ACKNOWLEDGMENTS**

We thank the Genome Modification at Harvard University for generating germ-line chimeras, Dr. Shiming Chen for providing rhodopsin-Cre mice on behalf of Dr. Jason Chen, and Dr. Evan Feinberg for valuable comments. This work was supported by the Gatsby Foundation, NIH (U24NS063931), and Collaborative Innovation Award #43667 from HHMI.

## **REFERENCES**

self-assembling fragments of green fluorescent protein. *Nat. Biotechnol.* 23, 102–107.

Chubykin, A. A., Atasoy, D., Etherton, M. R., Brose, N., Kavalali, E. T., Gibson, J. R., and Südhof, T. C. (2007). Activity-dependent validation of excitatory versus inhibitory synapses by neuroligin-1 versus neuroligin-2. *Neuron* 54, 919–931.

Dahlhaus, R., Hines, R. M., Eadie, B. D., Kannangara, T. S., Hines, D. J., Brown, C. E., Christie, B. R., and El-Husseini, A. (2010). Overexpression of the cell adhesion protein neuroligin-1 induces learning deficits and impairs synaptic plasticity by altering the ratio of excitation to inhibition in the hippocampus. *Hippocampus* 20, 305–322.

microscopy. *Nat. Methods* 9, 96–102.

specific synaptic connectivity. *Annu. Rev. Neurosci.* 33, 473–507.

Ueda, O., Suzuki, H., Tabuchi, K., Sawamoto, K., Okano, H., Noda, T., and Mori, K. (1999). A genetic approach to visualization of multisynaptic neural pathways using plant lectin transgene. *Neuron* 22, 33–41.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 14 January 2012; accepted: 03 February 2012; published online: 16 February 2012.*

*Citation: Yamagata M and Sanes JR (2012) Transgenic strategy for identifying synaptic connections in mice by fluorescence complementation (GRASP). Front. Mol. Neurosci. 5:18. doi: 10.3389/ fnmol.2012.00018*

*Copyright © 2012 Yamagata and Sanes. This is an open-access article distributed under the terms of the Creative Commons Attribution Non Commercial License, which permits non-commercial use, distribution, and reproduction in other forums, provided the original authors and source are credited.*


**8. Conclusions**

100 Otolaryngology

The ARIA document clearly states that immunotherapy (IT) is considered effective in AR, and that it is the only treatment that can change the natural course of the disease and

IT, both subcutaneous and sublingual, is an effective treatment for adults and children with severe AR that does not respond to conventional pharmacotherapy and allergen avoidance measures. The efficacy of IT depends on correct patient selection, the type of allergen and the product chosen for treatment. Each vaccine requires individual assessment before recommendation for routine use. In support of the conclusions of recent meta-analyses, data have provided further evidence for the efficacy of Sublingual immunotherapy (SLIT) at least

Due to the increased prevalence of AR, its impact on quality of life, its societal costs and the fact that it is a predisposing factor for asthma, new therapeutic options are being sought. Studies are being performed with anti-leukotrienes, anti-immunoglobulin E antibodies,

Schultz et al state that, in addition to the well established therapeutic guidelines in the treatment of rhinitis with antihistamines, corticosteroids, nasal decongestants, etc., there are an increasingly number of new therapeutic alternatives, such as anti-leukotrienes, antiimmunoglobulin E antibodies, phosphodiesterase inhibitors and intranasal heparin, as well as new specific immunotherapies (recombinant). There are promising results, but more

Bjermer and Diamant observed that, although inhaled corticosteroids are currently considered first-line drugs in the anti-inflammatory control of asthma and rhinitis, there are studies with anti-leukotrienes and anti-immunoglobulin E that are highly promising. Clinical trials are being performed with modified cytokines (Bjermer & Diamant, 2004).

Koreck et al reported on *low intensity UVB, UVA and visible light phototherapy* as treatment for AR (3 times per week for three weeks). The authors reported that there was a significant reduction in the number of eosinophils, ECP and IL-5 in the nasal lavage. They also found inhibition in the RBL-2H3 mediator release of basophils. Phototherapy is a new option in the treatment of immunologically mediated mucosal diseases, including allergic rhinitis (Koreck

Kirchhoff et al used the *H1-receptor antagonist dimethindene maleate* topically on patients with seasonal allergic rhinitis for 2 weeks and compared it with placebo. With this antagonist, they achieved statistically significantly better results in the quality of life questionnaires for

Unal et al investigated the potential benefits of the *toxin botulinum type A* on nasal symptoms of allergic rhinitis, and compared it to an isotonic saline solution as placebo. The results were significantly better in patients treated with botulinum toxin, especially in rhinorrhoea, nasal obstruction and sneezing. In selected cases, the injection of intranasal toxin botulinum

**7. Therapeutic developments in the treatment of allergic rhinitis (AR)**

phosphodiesterase inhibitors, and others, which seem to confirm promising results.

studies are needed to confirm these initial data (Schultz et al, 2003).

rhinitis than with placebo (Kirchhoff et al, 2003).

may help control allergic rhinitis symptoms (Unal et al, 2003).

prevent its evolution into asthma (Bousquet et al, 2008).

for grass pollen-induced (Table 2).

et al, 2005).

Considering the reviewed data, and unifying the above-mentioned opinions on the treatment of AR, we provide the following guidelines:


2.2.1. Testing Series

In this study, the tensile performance of carbon TRM was examined with respect to the reinforcement ratio of the textile, the addition of steel fibers at different volume fractions, and the prestressing force applied to the carbon textiles. The specific test schemes included two primary specimen series in which TRM composites that were reinforced with one or two textile layers were produced; control specimens with no textile layers were also produced.

The reinforcement ratios of the specimens that were produced with one and two layers of textile are 0.4% and 0.8%, respectively. The reinforcement ratio can be expressed as follows:

$$
\rho\_f = \frac{A\_f}{A\_c} \tag{1}
$$

where *Af* and *Ac* are the cross-sectional areas of the carbon yarns and TRM specimens, respectively.

The effects of steel fibers with different volume fractions (0.5%, 1%, and 2%) were studied in each series, and the effects of prestress on the mechanical behavior of the TRM composites were investigated by applying different prestressing forces to the one-layer (10% and 20% of the ultimate tensile strength of one-layer textile) and two-layer (15% of the ultimate tensile strength of two-layer textiles) specimens. Moreover, prestressed thin plates with 1% steel fibers added were researched to determine their effects on the mechanical behavior. The test specimens were named as follows: for example, P15C2S1 represents specimens with a prestress of 15% of the ultimate tensile strength of two-layer textiles (P15), a two-layer carbon textile arrangement (C2), and the addition of 1% (by volume) steel fibers (S1).


**7. References**

Transesterification in Supercritical Conditions 265

He, H., Sun, S., Wang, T. & Zhu, S. (2007a). Transesterification Kinetics of Soybean Oil for

He, H., Wang, T. & Zhu, S. (2007b). Continuous production of biodiesel fuel from vegetable

Hegel, P., Andreatta, A., Pereda, S., Bottini, S. & Brignole, E.A. (2008). High pressure phase

Hegel, P., Mabe, G., Pereda, S. & Brignole, E.A. (2007). Phase Transitions in a Biodiesel

Helwani, Z., Othman, M.R., Aziz, N., Kim, J. & Fernando, W.J.N. (2009). Solid

Ilham, Z. & Saka, S. (2009). Dimethyl carbonate as potential reactant in non-catalytic

Imahara, H., Minami, E., Hari, S. & Saka, S. (2008). Thermal stability of biodiesel in

Imahara, H., Xin, J. & Saka, S. (2009). Effect of CO2/N2 addition to supercritical methanol on

Khuwijitjaru, P., Fujii, T., Adachi, S., Kimura, Y. & Matsuno, R. (2004). Kinetics on the

Kiwjaroun, C., Tubtimdee, C. & Piumsomboon, P. (2009). LCA studies comparing biodiesel

Krammer, P. & Vogel, H. (2000). Hydrolysis of esters in subcritical and supercritical water. *The Journal of Supercritical Fluids*, Vol.16, No.3, pp. (189-206), ISSN 0896-8446 Kulkarni, M.G. & Dalai, A.K. (2006). Waste Cooking Oil An Economical Source for

Kusdiana, D. & Saka, S. (2001). Kinetics of transesterification in rapeseed oil to biodiesel fuel

Kusdiana, D. & Saka, S. (2004a). Catalytic effect of metal reactor in transesterification of

Kusdiana, D. & Saka, S. (2004b). Effects of water on biodiesel fuel production by

supercritical methanol. *Fuel*, Vol.87, No.1, pp. (1-6), ISSN 0016-2361

*Chemists' Society*, Vol.84, No.4, pp. (399-404), ISSN 0003-021X

*Fluid Phase Equilibria*, Vol.266, No.1-2, pp. (31-37), ISSN 0378-3812

Vol.46, No.19, pp. (6360-6365), ISSN 0888-5885

0016-2361

271), ISSN 1520-6106

1332), ISSN 0016-2361

pp. (1793-1796), ISSN 0960-8524

Vol.99, No.1, pp. (1-4), ISSN 1385-8947

pp. (2901-2913), ISSN 0888-5885

104), ISSN 0003-021X

ISSN 0960-8524

2361

*Production*, Vol.17, No.2, pp. (143-153), ISSN 0959-6526

Production of Biodiesel in Supercritical Methanol. *Journal of the American Oil*

oil using supercritical methanol process. *Fuel*, Vol.86, No.3, pp. (442-447), ISSN

equilibria of supercritical alcohols with triglycerides, fatty esters and cosolvents.

Reactor Using Supercritical Methanol. *Industrial and Engineering Chemistry Research*,

heterogeneous catalysts for transesterification of triglycerides with methanol: A review. *Applied Catalysis A: General*, Vol.363, No.1-2, pp. (1-10), ISSN 0926-860X Hoffmann, M.M. & Conradi, M.S. (1998). Are There Hydrogen Bonds in Supercritical

Methanol and Ethanol? *The Journal of Physical Chemistry B*, Vol.102, No.1, pp. (263-

biodiesel production by supercritical method. *Bioresource Technology*, Vol.100, No.5,

reactivities and fuel qualities in biodiesel production. *Fuel*, Vol.88, No.7, pp. (1329-

hydrolysis of fatty acid esters in subcritical water. *Chemical Engineering Journal*,

synthesized by conventional and supercritical methanol methods. *Journal of Cleaner*

Biodiesel:‰ A Review. *Industrial and Engineering Chemistry Research*, Vol.45, No.9,

as treated in supercritical methanol. *Fuel*, Vol.80, No.5, pp. (693-698), ISSN 0016-

vegetable oil. *Journal of the American Oil Chemists' Society*, Vol.81, No.1, pp. (103-

supercritical methanol treatment. *Bioresource Technology*, Vol.91, No.3, pp. (289-295),

Demirbas, A. (2002). Biodiesel from vegetable oils via transesterification in supercritical

Demirbas, A. (2007). Biodiesel from sunflower oil in supercritical methanol with calcium

Demirbas, A. (2008). Biodiesel from vegetable oils with MgO catalytic transesterification in

Deshpande, A., Anitescu, G., Rice, P.A. & Tavlarides, L.L. (2010). Supercritical biodiesel

Diaz, M.S., Espinosa, S. & Brignole, E.A. (2009). Model-Based Cost Minimization in

Espinosa, S., Fornari, T., Bottini, S.B. & Brignole, E.A. (2002). Phase equilibria in mixtures of

Furuta, S., Matsuhashi, H. & Arata, K. (2004). Biodiesel fuel production with solid superacid

Glišic, S., Lukic, I. & Skala, D. (2009). Biodiesel synthesis at high pressure and temperature:

Glišic, S. & Skala, D. (2009). The problems in design and detailed analyses of energy

Glišic, S.B. & Skala, D.U. (2010). Phase transition at subcritical and supercritical conditions of

Gui, M.M., Lee, K.T. & Bhatia, S. (2009). Supercritical ethanol technology for the production

Han, H., Cao, W. & Zhang, J. (2005). Preparation of biodiesel from soybean oil using

*Supercritical Fluids*, Vol.49, No.2, pp. (293-301), ISSN 0896-8446

*Journal of Supercritical Fluids*, Vol.23, No.2, pp. (91-102), ISSN 0896-8446 Fang, T., Shimoyama, Y., Abeta, T., Iwai, Y., Sasaki, M. & Goto, M. (2008). Phase equilibria

*Environmental Effects*, Vol.30, No.17, pp. (1645-1651), ISSN 1556-7036 Demirbas, A. (2009). Biodiesel from waste cooking oil via base-catalytic and supercritical

*Bioresource Technology*, Vol.101, No.6, pp. (1834-1843), ISSN 0960-8524 Diasakou, M., Louloudi, A. & Papayannakos, N. (1998). Kinetics of the non-catalytic

0196-8904

(923-927), ISSN 0196-8904

5595), ISSN 0887-0624

146), ISSN 0896-8446

80), ISSN 0896-8446

Vol.5, No.12, pp. (721-723), ISSN

No.24, pp. (6347-6354), ISSN 0960-8524

No.2, pp. (286-292), ISSN 0896-8446

(3148-3151), ISSN 1359-5113

8904

2361

methanol. *Energy Conversion and Management*, Vol.43, No.17, pp. (2349-2356), ISSN

oxide. *Energy Conversion and Management*, Vol.48, No.3, pp. (937-941), ISSN 0196-

supercritical methanol. *Energy Sources, Part A: Recovery, Utilization and*

methanol transesterification. *Energy Conversion and Management*, Vol.50, No.4, pp.

production and power cogeneration: Technical and economic feasibilities.

transesterification of soybean oil. *Fuel*, Vol.77, No.12, pp. (1297-1302), ISSN 0016-

Noncatalytic Biodiesel Production Plants. *Energy and Fuels*, Vol.23, No.11, pp. (5587-

fatty oils and derivatives with near critical fluids using the GC-EOS model. *The*

for the mixtures of supercritical methanol + C18 methyl esters and supercritical methanol + -tocopherol. *The Journal of Supercritical Fluids*, Vol.47, No.2, pp. (140-

catalysis in fixed bed reactor under atmospheric pressure. *Catalysis Communications*,

Analysis of energy consumption on industrial scale. *Bioresource Technology*, Vol.100,

consumption for biodiesel synthesis at supercritical conditions. *The Journal of*

triglycerides methanolysis. *The Journal of Supercritical Fluids*, Vol.54, No.1, pp. (71-

of biodiesel: Process optimization studies. *The Journal of Supercritical Fluids*, Vol.49,

supercritical methanol and CO2 as co-solvent. *Process Biochemistry*, Vol.40, No.9, pp.

Transesterification in Supercritical Conditions 267

Rathore, V. & Madras, G. (2007). Synthesis of biodiesel from edible and non-edible oils in

Saka, S. & Isayama, Y. (2009). A new process for catalyst-free production of biodiesel using supercritical methyl acetate. *Fuel*, Vol.88, No.7, pp. (1307-1313), ISSN 0016-2361 Saka, S. & Kusdiana, D. (2001). Biodiesel fuel from rapeseed oil as prepared in supercritical

Sawangkeaw, R., Bunyakiat, K. & Ngamprasertsith, S. (2007). Effect of co-solvents on

Sawangkeaw, R., Bunyakiat, K. & Ngamprasertsith, S. (2010). A review of laboratory-scale

*The Journal of Supercritical Fluids*, Vol.55, No.1, pp. (1-13), ISSN 0896-8446 Shimoyama, Y., Abeta, T. & Iwai, Y. (2008). Prediction of vapor-liquid equilibria for

Shimoyama, Y., Abeta, T., Zhao, L. & Iwai, Y. (2009). Measurement and calculation of vapor-

Song, E.-S., Lim, J.-w., Lee, H.-S. & Lee, Y.-W. (2008). Transesterification of RBD palm oil

Tan, K.T. & Lee, K.T. (2011). A review on supercritical fluids (SCF) technology in sustainable

Tan, K.T., Lee, K.T. & Mohamed, A.R. (2010a). Effects of free fatty acids, water content and

Tan, K.T., Lee, K.T. & Mohamed, A.R. (2010b). Optimization of supercritical dimethyl

van Kasteren, J.M.N. & Nisworo, A.P. (2007). A process model to estimate the cost of

glycerol carbonate. *Fuel*, Vol.89, No.12, pp. (3833-3839), ISSN 0016-2361 Tang, Z., Du, Z., Min, E., Gao, L., Jiang, T. & Han, B. (2006). Phase equilibria of methanol-

573 K. *Fluid Phase Equilibria*, Vol.284, No.1, pp. (64-69), ISSN 0378-3812 Silva, C., Weschenfelder, T.A., Rovani, S., Corazza, F.C., Corazza, M.L., Dariva, C. &

Vol.86, No.17-18, pp. (2650-2659), ISSN 0016-2361

methanol. *Fuel*, Vol.80, No.2, pp. (225-231), ISSN 0016-2361

*Chemistry*, Vol.9, No.6, pp. (679-685), ISSN 1463-9262

Vol.46, No.1, pp. (4-9), ISSN 0896-8446

Vol.46, No.16, pp. (5304-5309), ISSN 0888-5885

*Reviews*, Vol.15, No.5, pp. (2452-2456), ISSN 1364-0321

*Supercritical Fluids*, Vol.53, No.1-3, pp. (88-91), ISSN 0896-8446

(356-363), ISSN 0896-8446

No.1, pp. (8-11), ISSN 0378-3812

ISSN 0921-3449

8446

methanol. *The Journal of Supercritical Fluids*, Vol.56, No.1, pp. (56-63), ISSN 0896-

supercritical alcohols and enzymatic synthesis in supercritical carbon dioxide. *Fuel*,

production of biodiesel via transesterification in supercritical methanol. *Green*

research on lipid conversion to biodiesel with supercritical methanol (2001-2009).

supercritical alcohol + fatty acid ester systems by SRK equation of state with Wong-Sandler mixing rule based on COSMO theory. *The Journal of Supercritical Fluids*,

liquid equilibria for methanol + glycerol and ethanol + glycerol systems at 493-

Oliveira, J.V. (2007). Continuous Production of Fatty Acid Ethyl Esters from Soybean Oil in Compressed Ethanol. *Industrial and Engineering Chemistry Research*,

using supercritical methanol. *The Journal of Supercritical Fluids*, Vol.44, No.3, pp.

biodiesel production: Potential and challenges. *Renewable and Sustainable Energy*

co-solvent on biodiesel production by supercritical methanol reaction. *The Journal of*

carbonate (SCDMC) technology for the production of biodiesel and value-added

triolein system at elevated temperature and pressure. *Fluid Phase Equilibria*, Vol.239,

industrial scale biodiesel production from waste cooking oil by supercritical transesterification. *Resources, Conservation and Recycling*, Vol.50, No.4, pp. (442-458),

Lam, M.K., Lee, K.T. & Mohamed, A.R. (2010). Homogeneous, heterogeneous and

Lee, J.-S. & Saka, S. (2010). Biodiesel production by heterogeneous catalysts and supercritical

Lene, F., Knud, V.C. & Birgir, N. (2009). A review of the current state of biodiesel production

Levine, R.B., Pinnarat, T. & Savage, P.E. (2010). Biodiesel Production from Wet Algal

Lima, D.G., Soares, V.C.D., Ribeiro, E.B., Carvalho, D.A., Cardoso, É.C.V., Rassi, F.C.,

Liu, X., He, H., Wang, Y., Zhu, S. & Piao, X. (2008). Transesterification of soybean oil to

Madras, G., Kolluru, C. & Kumar, R. (2004). Synthesis of biodiesel in supercritical fluids.

Marchetti, J.M. & Errazu, A.F. (2008). Technoeconomic study of supercritical biodiesel

Marulanda, V.F., Anitescu, G. & Tavlarides, L.L. (2009). Biodiesel Fuels through a

Marulanda, V.F., Anitescu, G. & Tavlarides, L.L. (2010). Investigations on supercritical

Meher, L.C., Vidya Sagar, D. & Naik, S.N. (2006). Technical aspects of biodiesel production

Minami, E. & Saka, S. (2006). Kinetics of hydrolysis and methyl esterification for biodiesel

Pinnarat, T. & Savage, P.E. (2008). Assessment of noncatalytic biodiesel synthesis using

Poling, B.E., Prausnitz, J.M. & O'connell, J.P. (2001). *The properties of gases and liquids*. (5 ed).

Quesada-Medina, J. & Olivares-Carrillo, P. (2011). Evidence of thermal decomposition of

*Energy and Fuels*, Vol.24, No.9, pp. (5235-5243), ISSN 0887-0624

*Fuel*, Vol.83, No.14-15, pp. (2029-2033), ISSN 0016-2361

*and Fuels*, Vol.24, No.1, pp. (253-260), ISSN 0887-0624

No.3, pp. (248-268), ISSN 1364-0321

Vol.47, No.18, pp. (6801-6808), ISSN 0888-5885

McGraw-Hill, ISBN 7011-6822, New York.

(2479-2483), ISSN 0016-2361

0734-9750

0016-2361

ISSN 0196-8904

8446

pp. (1298-1315), ISSN 1097-0290

pp. (987-996), ISSN 0165-2370

8524

enzymatic catalysis for transesterification of high free fatty acid oil (waste cooking oil) to biodiesel: A review. *Biotechnology Advances*, Vol.28, No.4, pp. (500-518), ISSN

technologies. *Bioresource Technology*, Vol.101, No.19, pp. (7191-7200), ISSN 0960-

using enzymatic transesterification. *Biotechnology and Bioengineering*, Vol.102, No.5,

Biomass through in Situ Lipid Hydrolysis and Supercritical Transesterification.

Mundim, K.C., Rubim, J.C. & Suarez, P.A.Z. (2004). Diesel-like fuel obtained by pyrolysis of vegetable oils. *Journal of Analytical and Applied Pyrolysis*, Vol.71, No.2,

biodiesel using CaO as a solid base catalyst. *Fuel*, Vol.87, No.2, pp. (216-221), ISSN

production plant. *Energy Conversion and Management*, Vol.49, No.8, pp. (2160-2164),

Continuous Flow Process of Chicken Fat Supercritical Transesterification.  *The Journal of Supercritical Fluids*, Vol.54, No.1, pp. (53-60), ISSN 0896-

by transesterification--a review. *Renewable and Sustainable Energy Reviews*, Vol.10,

production in two-step supercritical methanol process. *Fuel*, Vol.85, No.17-18, pp.

supercritical reaction conditions. *Industrial and Engineering Chemistry Research*,

fatty acid methyl esters during the synthesis of biodiesel with supercritical

methanol. *The Journal of Supercritical Fluids*, Vol.56, No.1, pp. (56-63), ISSN 0896- 8446

**13**

*Colombia*

**Alternative Methods for**

Paula Mazo, Gloria Restrepo and Luis Rios

**Fatty Acid Alkyl-Esters Production:**

*Universidad de Antioquia. Grupo Procesos Fisicoquímicos Aplicados*

**Microwaves, Radio-Frequency and Ultrasound**

Biodiesel production is a very modern and technological area for researchers due to the relevance that it is winning every day because of the increase in the petroleum price and the

Biodiesel is a mixture of mono-alkyl esters of long chain fatty acids, is an alternative fuel made from renewable sources as vegetable oils and animal fats. It is biodegradable, nontoxic, show low emission profiles and also is beneficial environmentally. (Fangrui and

Biodiesel is quite similar to petroleum-derived diesel in its main characteristics such as cetane number, energy content, viscosity, and phase changes. Biodiesel contains no petroleum products, but it is compatible with conventional diesel and can be blended in any proportion with fossil-based diesel to create a stable biodiesel blend. Therefore, biodiesel has become one of the most common biofuels in the world (Lin et al., 2011). There are four primary techniques for biodiesel production: direct use and blending of raw oils, micro-

Direct use of vegetable oil and animal fats as combustible fuel is not suitable due to their high kinematic viscosity and low volatility. Furthermore, its long term use posed serious problems such as deposition, ring sticking and injector chocking in engine. Microemulsions with alcohols have been prepared to overcome the problem of high viscosity of vegetable oils. Another alternative way to produce biodiesel is through thermal cracking or pyrolysis. However, this process is rather complicated to operate and produce side products that have not commercial value. The most commonly used method for biodiesel production is transesterification (also known as alcoholysis) reaction in presence of a catalyst. Transesterification is the process of exchanging the alkoxy group of an ester compound with

Esterification is the sub category of trans-esterification. This requires two reactants, carboxylic acids (fatty acids) and alcohols. Esterification reactions are acid-catalyzed and proceed slowly in the absence of strong acids such as sulfuric, phosphoric, sulfonic-organic

The fatty acid methyl esters (FAME) are more used because of its facility of production, however, presents operating problems at low temperatures for its high content of saturated

emulsions, thermal cracking and trans-esterification (Siddiquee and Rohani, 2011).

**1. Introduction**

Milford, 1999).

environmental advantages (Mustafa, 2011).

another alcohol (Lam et al., 2010).

acids and hydrochloric acid (Vyas et al., 2010).