I.

INTRODUCTION
Polyglutamic acid (PGA) is a natural biopolymer made up of periodical units of both L-
glutamic acid, D-glutamic acid. This biopolymer is of special interest based on its
biodegradability, non-dangerous and non-immunogenic properties, and thus it has been
utilized effectively in many pharmaceutical applications. Among other novel applications, it
has the likelihood to be utilized for protein crystallization, as a delicate tissue follower and a
nonviral vector for safe quality conveyance. Therefore, PGA exhibited many therapeutic
applications as anticancer agent, drug delivery, biological Glues, biological control agent, and
in tissue Engineering. Investigations of PGA biosynthesis/production and learning of the
catalysts and functional biological actions will additionally increase the range of its
applications.

Large number of carboxyl groups along the

molecule chain of gamma PGA can form
hydrogen bonding in a molecule or between
different molecules. Thus it has high water
absorbability and moisture-retaining
capability. Thanks to its unique properties,
Gamma PGA can be used as thickener,
filmogen, humcctant, retarder, cosolvent,
binder and anti-freezer, therefore the application prospect of gamma PGA is promising.

Background
Poly--glutamic acid (-PGA) is an unusual anionic homopolyamide made from d-and l-
glutamic acid units connected through amide linkages between -amino and -carboxylic acid
groups. Based on the glutamate residues present, -PGA may be classified as -l-PGA (only
l-glutamic acid residues), -dPGA (only d-glutamic acid residues), and -LD-PGA (both l-
and d-glutamic acid residues). At present, there exist four methods for -PGA production:
chemical synthesis, peptide synthesis, biotransformation, and microbial fermentation.
Compared with other methods, microbial fermentation is the most cost-effective and has
numerous advantages, including inexpensive raw materials, minimal environmental pollution,
high natural product purity, and mild reaction conditions. Initially discovered in 1937 by
Bruckner and co-workers as part of the capsule of Bacillus anthracis, -PGA has since been
found in species from all three domains of life (archaea, bacteria, and eukaryotes). Most
commercial -PGA is currently produced via microbial fermentation from biomass. Unlike
most proteinaceous materials, -PGA is synthesized in a ribosome-independent manner; thus,
substances that inhibit protein translation (such as chloramphenicol) have no effect on the
production of -PGA. Furthermore, due to the -linkage of its component glutamate residues,
-PGA is resistant to proteases that cleave -amino linkages. More importantly, as a
biodegradable, water-soluble, edible, and non-toxic biopolymer, -PGA and its derivatives
can be used safely in a wide range of applications including as thickeners, humectants,
bitterness-relieving agents, cryoprotectants, sustained release materials, drug carriers, heavy
metal absorbers, and animal feed additives.
 II. HISTORICAL DEVELOPMENT
It is an extracellular polymer which is completely biodegradable and non-toxic to humans.
PGA is synthesized by several microorganisms, Bacillus species like B. licheniformis and B.
subtilis are generally utilized to produce PGA.

Bovarnick (1942) first showed that c-PGA is freely secreted into the medium after
fermentation of B. subtilis. Following this, considerable research has been carried out into the
production of c-PGA using B. subtilis. More strains of B. subtilis have been investigated for
the production of c-PGA than of B. licheniformis. c-PGA production was recently evaluated
by Scoffone et al. (2013). This was done by knocking out pgdS and ggt (genes for two
important c-PGA-degrading enzymes) in the laboratory strain B. subtilis 168. The effects of
single mutations (either pgdS deletion or ggt deletion) and double mutation (both pgdS and
ggt deletion) on c-PGA production were assessed. Results revealed that single mutations had
no significant improvement on c-PGA yield, whereas a twofold increase (.40 g l21 ) was
observed in the doublemutant strain compared with the WT strain. However, number average
molecular mass and weight average molecular mass of c-PGA produced by double-mutant
strains were lower compared with those of single-mutant strains and the WT strain. The pgdS
mutant strain presented the highest molecular mass (.36106 Da), probably due to reduced
endo-degradation activities.

PGA was first discovered in 1973 by Ivanovic and Buckner together with his co-workers in
the form of capsule in Bacillus anthracis. When a capsule of Bacillus anthracis was released
into the medium upon autoclaving.

Another naturally occurring source of Poly glutamic acid is

the mucilage of natto (A traditional fermented soybeans in
Japan), which contains a mixture of PGA and fraction
produced by Bacillus subtilis sawamura. Its widely known
that, PGA is produced mostly by Gram positive bacteria,
which include the genus Bacillus. However, it has also been
reported that at least one Gram-negative bacterium
(Fusobacterium nucleatum), some archaea and eukaryotes
have the ability to produce c-PGA . PGA has been reported
to be the major component in the viscous sticky mucilage of
Natto -a health food in Japan. Natto, has been in domestic and commercial use and consumed
for more than a thousand years, which is produced by steaming small soybeans and
fermenting them with starter culture of Bacillus subtilis.
PGA acts as a natural moisturizer and its hygroscopic and moisturizing effects were
comparable with Hyaluronic acid. It has extensive adsorbing properties and therefore used as
an absorbent molecules. Cross-linked g-PGA is one of the most useful Super Absorbent
Polymers.
The applications of g-PGA in skincare products were explored by Ben-Zur & Goldman
(2007). They found that c-PGA is a good hydrophilic humectant and has the ability to
increase the production of natural moisturizing factors such as urocanic acid, pyrrolidone
carboxylic acid and lactic acid compared with hyaluronic acid and soluble collagen. g-PGA
has also been shown to enhance the elasticity of the skin more than collagen and hyaluronic
acid as well as refresh and nourish the skin, making it smoother and more desirable.

Gamma-Poly glutamic acid is delivered in form of its sodium salts.

Polyglutamic acid has also been found in neurons of mice where it was covalently linked to
tubulin. Efforts have been made to insert the genes responsible for c-Polyglutamic acid
production into Escherichia coli and in plants such as tobacco to increase the knowledge
regarding the molecular mechanism of PGA production. It was reported that pgsA, pgsB and
pgsC genes were identified as essential for its production.In this review, we provide in brief a
comprehensive update on the production; mechanism of synthesis, properties and new
applications of bacterial PGA.

Recently, Cao et al. (2013) also cloned and coexpressed the pgsBCA genes and glutamate
racemase gene (racE/glr) into E. coli, and reported that the engineered E. coli strains had the
ability to synthesize c-PGA with excellent D-glutamate content due to racE integration.

Recent research has shown that PGA can be used in drug delivery for the controlled release
of paclitaxel. By conjugating paclitaxel, or other drugs to PGA, the drug compounds become
more stable and more water soluble. In addition, the conjugate can act as a drug depot for
sustained release, enabling prolonged drug exposure to tumor cells
 III. CLASSIFICATION

A. Based on Origin
The synthesis of polyglutamic acid (PGA) was repressed by exogenous glutamate in strains
of Bacillus licheniformis but not in strains of Bacillus subtilis, indicating a clear difference in
the regulation of synthesis of capsular slime in these two species. Although extracellular -
glutamyltranspeptidase (GGT) activity was always present in PGA-producing cultures of B.
licheniformis under various growth conditions, there was no correlation between the quantity
of PGA and enzyme activity. Moreover, the synthesis of PGA in the absence of detectable
GGT activity in B. subtilis S317 indicated that this enzyme was not involved in PGA
biosynthesis in this bacterium. Glutamate repression of PGA biosynthesis may offer a simple
means of preventing unwanted slime production in industrial fermentations using B.
licheniformis.

Polyglutamic acid (-PGA) is a biosynthesized polymer consisting of a polyamide structure

with pendant -carboxyl groups.1 Recently, an industrial synthetic process of -PGA has
been established using Bacillus subtilis F-2-01 (Scheme 1),2 and its reaction and application
attract much attention as biocompatible, functional materials. For example, esterification of
the -carboxyl group of -PGA has been examined with alkyl halides to afford the
corresponding -alkyl--PGA.3 -PGA is degraded with a protease bromelin, where the
degradation rate depends on the degree of esterification and alkyl chain length.4 -PGA
undergoes alkaline hydrolysis with sodium hydroxide to afford the -PGA fragments with
controlled molecular weights.5 -PGA may be applicable to polymer drugs because its benzyl
ester matrices gradually release biological active 4-fluorouracil.6 -PGA powder serves as an
initiator for cationic polymerization of N-vinylcarbazol and N-vinylpyrrolidone to form
surface-grafted polymers.7 Biologically synthesized -PGA contains a racemized unit with
the L- and D-glutamic acid unit ratio of 40 : 60.8 It is desirable to synthesize nonracemized -
PGA to obtain materials that would show highly controlled properties and functions. -PGA
cannot be synthesized by polycondensation of glutamic acid because intramolecular
cyclization predominantly proceeds to form a stable five-membered lactam, pyroglutamic
acid.

In contrast to peptide synthesis by ring-opening polymerization of five-membered amino

acid N-carboxyanhydride (NCA), the synthesis of -PGA from seven-membered glutamic
acid -NCA is not operative because the compound is so unstable that it easily releases
CO2 to afford pyroglutamate without forming -PGA.10Kajtr and Bruckner9 have
synthesized -PGA by polycondensation of glutamic acid dimers, followed by hydrolysis of
the ester group of the side chain. They have examined the solubility of -PGA, but the
spectroscopic and optical data have not been elucidated. The present article deals with the
synthesis of -methyl--PGA by polycondensation of glutamic acid (L,L)-, (L,D)-, and
(D,D)-dimers as well as reactions of the obtained polymers to form -PGA.

B. Based on monomer
The starting polyglutamic acid can be obtained from various sources. For example, the
starting polyglutamic acid can be obtained from a commercial source such as Sigma-Aldrich
Chemical Co. Alternatively, the starting polyglutamic acid can be synthesized. Suitable
methods for the synthesizing starting polyglutamic acid are known to those skilled in the art.
One method for synthesizing the starting polyglutamic acid is by reacting a glutamic ester
monomer with a suitable initiator. An example of a suitable reaction between a glutamic ester
monomer and an initiator is illustrated.

wherein R is an ester protecting group. Any ester protecting group known in the art or
previously mentioned herein can be used. In some embodiments R is Ci-Ci4 alkyl, C6-Cio
aryl, or C -Cj4 aralkyl. In some embodiments, R is benzyl, phenyl, t-butyl, isopropyl, ethyl,
or methyl.

For example, a benzyl ester glutamic acid N-carboxyanhydride can be reacted with an amine
initiator to produce a polyglutamic acid benzyl ester polymer, as illustrated. The amine
initiator can be triethyl amine (TEA). The reaction can be performed in dioxane at room
temperature. It possesses thermoplastic properties.
 IV. STRUCTURE, PROPERTIES AND APPLICATION
Structural characteristics of -PGA Generally, -PGA adopts five conformations; -helix, -
sheet, helix-to-random coil transition, random coil, and enveloped aggregate. The
conformation can be changed by altering environmental conditions such as pH, polymer
concentration, and ionic strength. For example, -PGA adopts a largely -helical
conformation at pH 7, but predominantly -sheet-based conformation at higher pH. The
enantiomeric composition also varies and can be manipulated through the extraction process
after fermentation. For example, -PGA containing only l or d enantiomers is soluble in
ethanol, whereas -PGA containing equimolar amounts of l and d precipitates in ethanol.
Manipulating the enantiomeric composition of -PGA to alter its properties is therefore
possible. The molecular mass of -PGA can also influence its properties and efficacy for
specific applications. Microbialderived -PGA generally has a relatively high molecular
weight (Mw ~105 8 106 Da), which can limit industrial applications due to high
viscosity, unmanageable rheology, and difficult modification. Therefore, polymers with
different molecular weights may be required for different purposes, and controlling the
molecular weight is of fundamental and practical importance for commercial development.
Recently, medium composition, alkaline hydrolysis, ultrasonic degradation, and microbial or
enzymatic degradation have all been used to alter the molecular weight of -PGA. Of these,
ultrasonic irradiation provides an interesting alternative to enzymatic hydrolysis and has been
proposed to reduce both the molecular weight and polydispersity of -PGA without
disturbing the chemical composition of the polymer.

Structure Properties Application

-PGA can exhibit different properties (conformational

states, enantiomeric properties and molecular mass).
biodegradable, non-toxic and non-immunogenic it has been used
properties successfully in the
Optimal skin hydration is a critical factor to beautiful food, medical and
and healthy skin. wastewater
nontoxic, edible, adhesive, film-forming, and moisture industries.
retention
Physical and Chemical Properties of PGA: g-PGA is water-soluble and anionic polymer,
however its free acid form (H+) is insoluble in water, whereas the salt forms like K+, Na+,
NH4+, Ca2+ and Mg2+ of g-polyglutamates are completely soluble in water. 11, 12 It is
completely biodegradable and nontoxic to humans 8 and thus can be used as biological
glues. 16, 17 It acts as a natural moisturizer 14 and its hygroscopic and moisturizing effects
were comparable with Hyaluronic acid (HA). 18 It has extensive adsorbing properties and
therefore used as an absorbent molecules. 14 Cross-linked g-PGA is one of the most useful
Super Absorbent Polymers (SAP).

The molecular weight of PGA ranges from 100 KDa to 2,500 KDa (Kilo Daltons). The
ultimate molecular weight of PGA is dependent on many factors, such as an increase in
fermentation time will decrease the molecular weight of PGA because of the production of an
enzyme that catalyzes the hydrolytic breakdown of PGA. 12, 19

The other factors like pH, aeration, agitation, ionic strength etc., also affect the molecular
weight of PGA which is produced during fermentation.6, 82Molecular weight variation of
PGA allows various kinds of modifications and thus can have broad range of
applications. 18 The reported size of PGA filaments from B. Subtilis varies from 160 KDa to
1500 KDa (about 124011630 Glutamate residues). 23, 24

Poly-g-glutamate may assume quite a few different structures. The structure of PGA has been
predicted and proposed by eliciting the peptides of 10 or 20 glutamate molecules. In aqueous
solution, the conformational model consists of a left-handed helix which is stabilized by
intra-molecular hydrogen bonds has been reported. 20

Another study of PGA, purified from Bacillus licheniformis showed that its conformation is
flexible, depending on the PGA concentration and pH of the solution. 21 At low
concentration (0.1% w/v) when the pH is below 7.0, PGA adopts a conformation based
largely on -helices, whereas a -sheet-based conformation predominates at pH>7.0. The -
sheet conformation exposes the negative charges of PGA most efficiently because at higher
pH, i.e., at low H+ concentration, the Glutamic acid tends to give or release charges and it has
been proved that at higher pH, PGA possess sheet conformations. 21 It has been recently
reported that an unordered conformation occurred in circular dichroism experiments, but the
proposed mechanism was not completely elucidated and the test pH was also not validated.
 Applications of -PGA and its derivatives

Applications of -PGA Due to being water soluble, biodegradable, edible, and non-toxic, -
PGA and its derivatives have been applied in a broad range of industrial fields, including
food, cosmetics, agriculture, medicine, and bioremediation.
 Food industry
-PGA is used in the food industry, specifically in naturally occurring mucilage of natto
(fermented soybeans), but also as a food supplement, osteoporosis-preventing agent, texture
enhancer, cryoprotectant, and oil-reducing agent (Table 4). As a cryoprotectant, -PGA
enhances the viability of probiotic bacteria during freeze-drying, and -PGA was found to
protect Lactobacillus paracasei more effectively than sucrose, trehalose, or sorbitol [11, 67].
More importantly, as a food supplement, -PGA could effectively increase the bioavailability
of calcium by increasing its solubility and intestinal absorption, which decreased bone loss in
humans [68].

Medicine
As shown in Table 2, -PGA and its derivatives have been exploited as metal chelators and
drug carriers, and used in tissue engineering and as a biological adhesive in medicine. As a
drug delivery agent, the molecular mass of -PGA was the decisive factor determining the
drug delivery properties, including controlling the rate of drug release. For example, a -PGA
molecular weight of ~36 104 Da was used to produce paclitaxel poliglumex (a conjugate
of -PGA and paclitaxel), and this significantly improved both the safety and efficiency of
the drug (compared with standard paclitaxel) by enhancing its pharmacokinetic profile and
water solubility. Furthermore, this improved tumor selectivity via enhanced accumulation and
retention in tumor tissue [69].

Wastewater treatment
Due to its non-toxic and biodegradable properties, -PGA offers an eco-friendly
alternative for wastewater treatment. -PGA with a molecular weight of ~5.8 6.2 106 Da
appears to be superior to many conventional flocculants used in wastewater treatment plants
operating downstream of food processing fermentation processes [70]. More interestingly, -
PGA with a molecular weight of 9.9 105 Da could effectively remove 98 % of basic dyes
from aqueous solution at pH 1 and could then be re-used [71].

Other applications
-PGA has also been explored for use in cosmetics as a hydrophilic humectant to
increase the production of natural moisturizing agents such as urocanic acid, pyrrolidone
carboxylic acid, and lactic acid [72]. Many other applications of -PGA likely remain to be
discovered.

V. MANUFACTURING AND FABRICATION

One of the major challenges of making c-PGA usage applicable in industry is its cost.
According to Sung et al. (2005b), c-PGA is several tens to hundreds fold more expensive
than the conventional materials it is envisioned to replace. Reducing the cost of production is
the only foreseeable solution to this problem. Designing mass production systems for c-PGA
would be a major step towards a potent solution. To achieve this, one needs to obtain
thorough knowledge of how different factors affect the yield of production. Information
about genes and enzymes involved in c-PGA production would certainly help in manipulating
organisms for more efficient production of c-PGA. The past 15 years have seen a rise in
research in this direction (Ashiuchi & Misono, 2002; Sung et al., 2005b; Candela & Fouet,
2006; Buescher & Margaritis, 2007) and genes that play a role in every step of c-PGA
production have been identified. This section attempts to provide an understanding of the
current knowledge of the mechanism of c-PGA synthesis. A biosynthetic pathway for the
production of c-PGA has been proposed (Fig. 2). L-Glutamic acid units that make up c-PGA
can be derived from two sources. They can be obtained via the glutamic acid biosynthetic
pathway either exogenously or endogenously. Endogenous production of L-glutamic acid
requires conversion of a carbon source via acetyl-CoA and TCA cycle intermediates. a-
Ketoglutaric acid from the TCA cycle serves as a direct precursor of glutamic acid synthesis
(Ko & Gross, 1998; Rehm, 2009). Exogenous L-glutamic acid can be converted to
Lglutamine with the help of the enzyme glutamine synthase. L-Glutamine is a precursor of c-
PGA as well. The process of c-PGA synthesis can be seen to have four distinct stages: cPGA
racemization, c-PGA polymerization, c-PGA regulation and c-PGA degradation.
 -PGA polymerization
In the case of B. anthracis, the genes involved in -PGA synthesis lie on a large plasmid, as
opposed to other Bacillus species where the genes are present on the chromosome (Ashiuchi
et al., 2001a; Shih & Van, 2001). When c-PGA is surface associated (as in the case of B.
anthracis), cap (capsule) genes are required for its production. However, for c-PGA that is
released, the pgs (polyglutamate synthase) genes come into action (Candela & Fouet, 2006).
Both the cap and pgs gene sets have at least four genes: capB, C, A and E or pgsB, C, A and
E (Fig. 3). The pgsBCA genes of B. subtilis IFO3336 (B. natto) are homologous to the
capBCA genes of B. anthracis (Makino et al., 1989; Shih & Van, 2001). pgsBCA has been
identified as the sole machinery responsible for c-PGA synthesis in Bacillus species. To
prove this, Sung et al. (2005b)disrupted pgsBCA genes in B. subtilis (chungkookjang)
creating pgsBCA-null mutants incapable of c-PGA production. In contrast, Urushibata et al.
(2002a) are of the opinion that only pgsB and
pgsC are required for the production of c-PGA.
The role of pgsE in the production of c-PGA is
dispensable since pgsB, pgsC and pgsAA at high
concentrations were able to form a complex
capable of producing c-PGA even in the absence
of pgsE (Candela et al., 2005). On the contrary,
Yamashiro et al. (2011) found pgsE to be
essential in c-PGA production as its introduction
into the production medium in the presence of
Zn2+ tripled the production rate of c-PGA from
B. subtilis (chungkookjang). Candela et al.
(2005) showed that CapE (a 47 aa peptide) is
also responsible and essential for the production
of c-PGA as it appeared to interact with CapA.
The unique membrane-bound PgsBCA complex
is highly unstable and hydrophobic, which
makes isolation of this complex challenging.
The mechanism of polymerization has been
shown to be dependent on ATP. The phosphoryl group of ATP is first transferred to a
terminal carboxyl group of elongated cPGA through substrate-dependent ATP hydrolysis
(Sung et al., 2005b). Then, due to a nucleophilic attack of an amino group of glutamic acid on
the phosphorylated carboxyl group, an amide linkage is formed. This reaction continues to
polymerize c-PGA at the active site of the synthase complex (PgsBCA). PgsB and PgsC
together form most parts of the complexs catalytic site, whereas PgsA seems to remove the
elongated chain from the active site so that the next monomer can be added and may also be
involved in transporting c-PGA (Ashiuchi et al., 2001b; Urushibata et al., 2002a). Activity of
PgsBCA was found to be dependent on Mg2+ (Ashiuchi et al., 2004). Transportation of c-
PGA outside the cell could be facilitated by a less compact cell membrane with shorter
phospholipids (Buescher & Margaritis, 2007).
 -PGA racemization
Generally, -PGA is synthesized from d- or l-glutamate alone, or from both l and d
enantiomers together [19, 20]. However, to incorporate d-glutamate into the growing l-chain,
l-glutamate (exogenous or endogenous) is first converted into d-glutamate by a racemization
reaction. In B. subtilis, two homologs of the glutamate racemase gene (racE/glr and yrpC)
have been identified, and glr is essential for converting l-glutamate into d-glutamate for the
synthesis of -PGA [21]. Interestingly, RacE and yrpC are cytosolic enzymes with a high
selectivity for glutamate and a preference for the l-form, but neither are responsible for the
synthesis of -PGA [22]. The functions of these enzymes remains unknown [22, 23].
-PGA polymerization As shown in Fig. 2, polyglutamate synthase (pgs) is encoded by four
genes (pgsB, C, A, and E) and their homologs in Bacillus species are ywsC, ywtAB, and
capBCA [1, 24]. Recently, pgsBCA was identified as the sole machinery responsible for
polymerizing -PGA at the active site of the synthase complex (PgsBCA) in an ATP-
dependent reaction [25]. PgsB and PgsC form the main parts of the catalytic site, whereas
PgsA removes the elongated chain from the active site, which is necessary for addition of the
next monomer and transporting -PGA through the compact cell membrane [8]. The role of
pgsE in the production of -PGA was found to be dispensable, and high concentrations of
pgsB, pgsC, and pgsA were able to form -PGA in the absence of pgsE. However, other
 researchers found that pgsE was essential for -PGA production in the presence of Zn2+ in B.
subtilis. This may be because the unique membrane-bound PgsBCA complex is highly
unstable and hydrophobic, which could affect its isolation.

Fabrication of poly(-glutamic acid) monolith by thermally induced phase separation

and its application.
Monoliths are functional porous materials with a three-dimensional continuous
interconnected pore structure in a single piece. A monolith with uniform shape based on
poly(-glutamic acid) (PGA) has been prepared via a thermally induced phase separation
technique using a mixture of dimethyl sulfoxide, water, and ethanol as solvent. The
morphology of the obtained monolith was observed by scanning electron microscopy and the
surface area of the monolith was evaluated by the Brunauer Emmett Teller method. The
effects of fabrication parameters such as the concentration and molecular mass of PGA and
the solvent composition have been systematically investigated. The PGA monolith was cross-
linked with hexamethylene diisocyanate to produce the water-insoluble monolith. The
addition of sodium chloride to the phase separation solvent affected the properties of the
cross-linked monolith. The swelling ratio of the cross-linked monolith toward aqueous
solutions depended on the buffer pH as well as the monolith fabrication condition. Copper(II)
ion was efficiently adsorbed on the cross-linked PGA monolith, and the obtained copper-
immobilized monolith showed strong antibacterial activity for Escherichia coli. By
combination of the characteristic properties of PGA (e.g., high biocompatibility and
biodegradability) and the unique features of monoliths (e.g., through-pore structure, large
surface area, and high porosity with small pore size), the PGA monolith possesses large
potentials for various industrial applications in the biomedical, environmental, analytical, and
separation fields.

VI. MODIFICATION OF PROPERTIES

Additives Effects Original Properties New Application
Maximize the ability of water-soluble and anionic Poly Glutamic acid used in
Pt Agents
best drug delivery system polymer anti-cancer drug

To improve the The degradation rate is Used as biodegradable drug

Aspartic acid
uncontrolled degradation fast. carrier
rate

VII. ENVIRONMENT IMPACT

Soil
Recently, with numerous environmental problems being caused by chemical fertilizer
overuse, agricultural practices are shifting toward the development of environmentally
friendly N fertilizers. In this study, pot and field experiments were simultaneously conducted
to investigate the effect of poly(y-glutamic acid) (-PGA) on the yield, N use efficiency, and
soil microenvironment of wheat. Our study demonstrates a statistically significant increase in
winter wheat, number of tillers, seed number per spike, yield, soil microbial biomass N
(SMBN), and soil enzymes after -PGA application. The highest grain yield of 7435.69
55.91 kg ha-1 was obtained after -PGA application in the field experiment, which was
7.17% higher than the urea control. The N recovery efficiency increased by 11.81%-14.00%
and 11.30%-11.38% after the application of -PGA in pot and field experiments, respectively.
More mineral nitrogen in soil was immobilized by the microbes after -PGA application at
the early growth stage of wheat. The immobilized nitrogen was gradually released at the late
growth stage. The results demonstrate that -PGA can be used as a fertilizer synergist.
Modern agriculture now feeds 6 billion people in the global world. To meet the recent
growing demand for food, investments on agriculture are increasing. Consequently, synthetic
fertilizer application is also increasing (Thakur et al, 2013). Globally, fertilizer nitrogen (N)
application has increased rapidly over the past several decades, from 32 million tons in 1970
to around about 100 million tons in 2010; it is expected to increase to 130-150 million
tons/year by 2050 (Matson et al, 1998). However, the overuse of chemical fertilizers in
farmlands is a major contributor to environmental problems such as ground water
eutrophication, nitrate pollution, phosphate pollution, and others (Datta et al., 2010;
Mahmoud and Abd EL-Kader, 2012). Due to increasing human health concerns and pressure
in meeting stringent consumer standards, agricultural practices are shifting toward the
development of nitrogen (N) fertilizers that are environmentally friendly, possess high N
utilization efficiency, and can sustain crop production. Several strategies have been proposed
to reduce N losses, such as the use of urease and nitrification inhibitors or coated fertilizers
(Bremner, 1995; Han et al, 2008; Khalil et al., 2009). However, all these fertilizer
technologies have disadvantages. For example, coated fertilizer is too expensive for wide
agricultural use. Several nitrification inhibitors are also not easily available and likely to be
toxic to animals and other microorganisms (Iizumi et al., 1998). Therefore, an
environmentally friendly fertilizer synergist is required.
The significant increase in the yield after -PGA application can be due to the changes
induced by -PGA in the soil biological properties (including soil enzyme activities and
biomass size) (Liu et al., 2013). More mineral N in soil was immobilized by the microbes
after -PGA application at the early growth stage of wheat, and the immobilized N was
gradually released at the late jointing and booting stages. The size of the microbial biomass N
pool, which plays a key role in the N cycle (Bristow and Jarvis, 1991; Lovell and Jarvis,
1996), was significantly affected by -PGA application (Figure 2(c)). The amount of N
lost with -PGA treatment is lower than that in the no--PGA control. The amount of soil N
available for plant growth may be increased by -PGA application. Some other possible
reasons are as follows. First, previous research showed that the dry weight of shoots and
roots, as well as the root-to-shoot ratio of cucumber seedlings can be increased by -PGA
application (Wang et al., 2008). Physiologically, differences among N acquisition can be
attributed to the root size (Marschener, 1998; Kim et al., 2001; Liu et al., 2009), and wheat
root can be stimulated by providing -PGA both in the pot and field experiments.
Thus, the the roots of plants may have a higher capacity for nutrient uptake. Second, the
assimilation of N plays an important role in plant growth and development. The first step of
the incorporation of inorganic N into organic nitrogenous compounds via the glutamine
synthetase/glutamate synthase assimilatory pathway is likely to be a major checkpoint for
controlling the assimilation of plant N (Cai et al., 2009) The improvement in the yield and N
uptake of wheat after -PGA application may be due to the increase in the activities of
glutamine synthetase and glutamate synthase by -PGA. Third, -PGA possibly affects the
rates of ammonium ion uptake in plant roots. The mechanism by which plants are stimulated
by -PGA requires further study. To study the effect of the form of added -PGA on wheat
productivity and soil microbes, two fertilizer forms of -PGA were designed in the pot
experiment, namely, PGAU1 and PGAU2. PGAU1 was urea mixed with purified -PGA.
PGAU2 was urea coated with purified -PGA. The addition level of -PGA in the two
fertilizers was the same. PGAU2 was found to work better than PGAU1, which can be
attributed to -PGA being a polyanionic biopolymer (Lin et al., 2006). Urea coated with
purified -PGA highly adsorbs ammonium N, which can reduce N loss.
These effects can be attributed to the fertilizer synergist activity of -PGA, which may
contribute to decreased excessive chemical fertilizer use and environmental pollution.

Cold Stress
Cold stress adversely affects plant growth and development. Poly(-glutamic acid) (-PGA)
is a potential plant growth regulator that may be an effective cryoprotectant that prevents
crops from damage during cold weather. In this study, the effects of -PGA on the
physiological responses of rape seedlings subject to cold stress were investigated using
hydroponic experiments. We determined that the malondialdehyde content was decreased by
33.4% and the proline content was increased by 62.5% by -PGA after 144 h under cold
stress. Antioxidant enzymes activities were also evidently enhanced after treatment with -
PGA. These responses counteracted increases in the fresh weight and chlorophyll content of
rape seedlings, which increased by 24.5 and 50.9%, respectively, after 144 h, which meant
that growth inhibition caused by cold was mitigated by -PGA. Our results also showed that
-PGA also regulated Ca2+ concentrations in the cytoplasm and calcium-dependent protein
kinases, which are associated with cold resistance. In conclusion, we suggest that the
Ca2+/CPKs signal pathway is involved in the -PGA-mediated enhancement of cold
resistance in rape seedlings.

Calcium Absorption in Human

Poly-gamma-glutamic acid (PGA) increases calcium (Ca) solubility in vitro and in vivo, and
is associated with reduced bone loss in post-menopausal Japanese women. This study is the
first to examine the effect of PGA on Ca absorption in humans. :
A single-blind, randomized, crossover study with a 3-4 week wash-out was performed to
determine the effect of PGA (80.6% glutamic acids) on Ca absorption measured by the
double stable isotope method. Twenty-four healthy, non-smoking, postmenopausal women
(mean age: 56.4 +/- SE 0.9) were given 200 g of orange juice containing 200 mg Ca as Ca-44
enriched CaCO(3), with or without 60 mg of PGA, after an overnight fast. The two tests were
separated by 3-4 weeks. An intravenous injection of Ca-42 (CaCl(2) solution) was given 30
min after consuming the drink and a complete urine collection carried out from 24-48 h post-
dosing. Ca absorption was calculated from the Ca isotope ratios measured by thermal
ionization quadrupole mass spectrometry (TIQMS).
Mean Ca absorption with PGA was significantly higher (P < 0.01) than without PGA, 39.1
(SE 1.6) % and 34.6 (SE 1.9) %, respectively. The effect of PGA on increasing Ca absorption
was more marked in a sub-group of subjects whose baseline Ca absorption (without PGA)
was lower than the population mean value. Postmenopausal women who received a single
dose of PGA increased their intestinal Ca absorption particularly those individuals with lower
basal absorptive capacity.

VIII. MATERIALS DEGRADATION AND PRODUCT FAILURE

c-PGA degradation A number of enzymes have been shown to be associated with the
degradation of c-PGA. c-Glutamyl-transpeptidases (GGT) are enzymes that breakdown the
transfer of a cglutamyl group from a donor species to acceptor (peptides and amino acids)
species by developing an intermediate c-glutamyl enzyme in a transpeptidation reaction
(Morelli et al., 2014). The enzyme has the ability to perform exohydrolase activity towards c-
PGA, and the released glutamic acid is used by the bacterium as a source of carbon and
nitrogen (Kimura et al., 2004; Morelli et al., 2014). pgdS (also pgsS) is located downstream
and in the same orientation as the pgsBCA operon (Ashiuchi et al., 2003a; Suzuki & Tahara,
2003; Buescher & Margaritis, 2007). It is known to encode the enzyme c-glutamyl-hydrolase
(PgsS), which is responsible for c-PGA degradation and cleaves cPGA between two D-
glutamates (Ashiuchi et al., 2003b; Candela & Fouet, 2006). CapD is a part of the GGT
family and is required for the covalent anchoring of c-PGA to the peptidoglycan as well as
acting as a depolymerase (Candela & Fouet, 2005). CapD cleaves the c-PGA and transfers it
to either an acceptor molecule or H2O, resulting in transpeptidation or hydrolysis,
respectively (Candela et al., 2014). As c-PGA can be either anchored to the bacterial surface
or released, whilst CapD catalyses the anchorage of c-PGA to peptidoglycan, PgsS catalyses
the release of c-PGA (Candela & Fouet, 2006). The strains that have pgsBCA, but do not
produce c-PGA, do so because the genes are not translated and not because an inactive gene
product is produced (Urushibata et al., 2002b). Yao et al. (2009) investigated the presence
and activity of cPGA depolymerase enzyme in B. subtilis NX-2, which is responsible for the
depolymerization of c-PGA in batch culture. The enzyme was seen to be active
extracellularly in the culture and was shown to be an endo-hydrolase. The gene encoding the
enzyme was ywtD (pgsS). The YwtD protein was obtained in purified form after the gene
was cloned and expressed in E. coli. The enzyme was active over a temperature range of 30
40 uC and a pH range of 58. At the optimal pH and temperature (5 and 30 uC respectively),
a reduction in c-PGA molecular mass was observed from 1000 to 20 kDa and dispersity
decreased as a function of depolymerization time. The enzyme was also seen to be active
extracellularly during the late stationary phase. The study demonstrated a mild method for
controlled reduction of molecular mass, and could be used as a better alternative to physical
and chemical methods of degradation.
 Have you ever tasted the Japanese speciality natto? If you have, you might like to
know that it consists largely of poly- -glutamate (PGA).
More than 20 unrelated proteins can form amyloid fibrils in vivo which are related to various
diseases, such as Alzheimers disease, prion disease, and systematic amyloidosis. Amyloid
fibrils are an ordered protein aggregate with a lamellar cross- structure. Enhancing amyloid
clearance is one of the targets of the therapy of these amyloid-related diseases. Although
there is debate on whether the toxicity is due to amyloids or their precursors, research on the
degradation of amyloids may help prevent or alleviate these diseases. In this study, we
explored the amyloid-degrading ability of nattokinase, a fibrinolytic subtilisin-like serine
protease, and determined the optimal conditions for amyloid hydrolysis. This ability is shared
by proteinase K and subtilisin Carlsberg, but not by trypsin or plasmin.

IX. TECHNOLOGICAL ADVANCEMNT OF POLYGLUTAMIC ACID

Polymeric Micelles: New Targeted Drug Delivery Systems for Cancer Therapy
July 25, 2017

Polymeric micelles are an

important class of targeted drug
delivery systems that act as
carriers of chemotherapeutic
agents. A recent review
summarized the research being
carried out in the development and
use of polymeric micelles and
micelle-forming drug-polymer
conjugates for cancer therapy.
A common problem in cancer treatment is the untargeted delivery of highly toxic drugs,
which kills healthy cells in addition to cancer cells and results in serious side effects. Another
notable problem that compounds the toxic side effects of these drugs is their insolubility in
water and blood, which reduces their circulation time in the blood and necessitates the
administration of larger and more frequent doses of the drug.

For the reasons cited above, a targeted drug delivery system that selectively delivers a drug to
the tumor site and leaves healthy tissue unaffected is a prized goal for many researchers. A
review published in AAPS PharmSciTech in 2014 provided a comprehensive overview of
ongoing research in this area, some of which is summarized below.

Many chemotherapeutic drugs are insoluble in water and blood, which reduces their effective
availability at the site of the tumor. However, these drugs are oil-loving or soluble in oils,
fats, detergents, and compounds such as those found in cell membranes. This makes micelles
prime candidates for carrying these drugs to specific sites in the body where drug action is
required. Micelles are aggregates that are formed when molecules or chemical compounds
that have both water-loving (called hydrophilic) and water-repelling (called
hydrophobic) segments are added to water or water-based solutions. Detergents are examples
of micelle forming compounds, and as for soap bubblesyes, they are micelles. When added
to water, the detergent molecules aggregate in a manner that allows their hydrophilic
segments to interact with water while their hydrophobic or oily segments are buried in the
interior of the aggregate. It is now easily seen how micelles can serve as carriers for oil-
loving or hydrophobic drugs.

Polymeric micelles, commonly used as drug carriers, are generally formed from molecules
called block co-polymers, which have very large hydrophilic and hydrophobic segments. The
hydrophilic segments of polymeric micelles for drug delivery generally consist of
polyethylene glycol (PEG) with a molecular weight of 215 kilodaltons. Their hydrophobic
segments are prepared from polyesters such as poly(lactic acid) (PLA) and polyamides such
as poly (L-lysine) (PLL) and poly (beta-amino ester). Genexol-PM is an example of a
polymeric micelle formulation containing the antitumor drug paclitaxel that is available in the
market for the treatment of breast cancer, non small cell lung cancer (NSCLC), and ovarian
cancer. Additionally, its efficacy in treating gynaecologic cancer in combination with
carboplatin is currently being tested in a phase I clinical trial.
Researchers have developed different types of polymeric micelles with an improved capacity
to load drugs into the micelle interior. This was achieved by incorporating chemical groups
that can interact with the drugs in the interior of the micelle. This strategy has been
successfully used to create micelles with improved loading capacities for a number of drugs
including doxorubicin, camptothecin, and paclitaxel. However, more tests for efficacy and
safety need to be carried out before these formulations reach the clinical trial stage.

In a novel strategy, researchers are have tried to use micelle forming conjugates of
hydrophobic drugs and hydrophilic polymers to load a second drug, which would be carried
in the micelle interior. Loading of the antitumor drug cisplatin on the polyglutamic acid and
paclitaxel conjugate (paclitaxel poliglumex) is an example of this strategy being successfully
deployed to improve efficacy while lowering toxicity.

Another example is the PEG conjugate with vitamin E called D--tocopheryl polyethylene
glycol (PEG) 1,000 succinate (TPGS). TPGS has been approved for use by the United States
Food and Drug Administration as a pharmaceutical adjuvant. TPGS helps overcome
multidrug resistance in cancer and increases the availability of orally taken cancer drugs at
tumor sites. TPGS has been conjugated to doxorubicin, and this formulation is eliminated
more slowly and has fewer side effects when administered in rats.

Similarly, embelin, a small molecule compound isolated from a Japanese herb, has been
conjugated with PEG, and PEG-embelin formulations with paclitaxel have been shown to
possess superior antitumor activity compared to taxol when administered to mice with breast
or prostate cancer. Embelin is known to bind and inhibit a protein called XIAP, which is
expressed in large quantities in tumor cells but not normal cells. Therefore, drug formulations
containing embelin are expected to accumulate at tumor sites while avoiding healthy tissues,
improving both drug efficacy and safety. In a similar vein, another small molecule compound
called S-trans,trans-farnesylthiosalicylic acid, which binds to and inhibits the activity of the
tumor-causing protein Ras, has been conjugated to PEG and when used in combination with
paclitaxel shows significantly higher anticancer activity than taxol in mice with breast cancer.

In other experiments, researchers are attempting to conjugate PEG or other polymers to

curcumin, a compound isolated from turmeric with known antitumor activity against
melanomas and cancers of the prostate, colon, breast, liver, kidney, and lymphoid and
myeloid tissues, to improve its solubility and availability at tumor sites. These are but a few
examples. Further improvements in the methods of conjugating drugs to polymeric carriers
and identifying new polymeric carriers that do not trigger allergic or hypersensitive responses
are expected to pave the way for exciting new targeted drug delivery systems for treating
cancer and other chronic diseases.

New Research: Non-Hodgkin Lymphoma Pipeline Review H1 2017 Industry Research

Toward a Cure, And Immune-Based and Gene Therapies in Development

JUNE 13, 2017

Non-Hodgkin Lymphoma Market Pipeline Review, H1 2017, latest research study provides
in depth analysis on Cellular Tumor Antigen P53 (Tumor Suppressor P53 or Antigen NY-
CO-13) targeted pipeline therapeutics. Non-Hodgkin Lymphoma therapeutics industry report
provides comprehensive information on the therapeutics under development for Non-
Hodgkin Lymphoma, complete with analysis by stage of development, drug target,
mechanism of action (MoA), route of administration (RoA) and molecule type. The report
also covers the descriptive pharmacological action of the therapeutics, its complete research
and development history and latest news and press releases. Non-Hodgkin Lymphoma
(Oncology) pipeline guide helps in identifying and tracking emerging players in the market
and their portfolios, enhances decision making capabilities and helps to create effective
counter strategies to gain competitive advantage.

Leading key players involved in Non-Hodgkin Lymphoma Market Pipeline Review, H1

2017: AB Science SA, AbbVie Inc, ACEA Biosciences Inc, Aileron Therapeutics Inc,
and Others.

Non-Hodgkin lymphoma (also known as non-Hodgkin lymphoma, NHL, or sometimes just

lymphoma) is a cancer that starts in cells called lymphocytes, which are part of the bodys
immune system. Symptoms depend on what area of the body is affected by the cancer and
how fast the cancer is growing.

The report helps in identifying and tracking emerging players in the market and their
portfolios, enhances decision making capabilities and helps to create effective counter
strategies to gain competitive advantage. Key Topics Covered are Introduction, Overview,
Therapeutics Development, Pipeline Products for Non-Hodgkin Lymphoma Overview,
Pipeline Products for Non-Hodgkin Lymphoma Comparative Analysis, Non-Hodgkin
Lymphoma Therapeutics under Development by Companies, Non-Hodgkin Lymphoma
Therapeutics under Investigation by Universities/Institutes, Products Glance, Discontinued
Products, Featured News & Press Releases, And Continue.

Scope of Non-Hodgkin Lymphoma Market Pipeline Review Report-

The report provides a snapshot of the global therapeutic landscape of Neuropathic Pain, The
report reviews pipeline therapeutics for Non-Hodgkin Lymphoma Industry by companies and
universities/research institutes based on information derived from company and industry-
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ranging from pre-registration till discovery and undisclosed stages, The report features
descriptive drug profiles for the pipeline products which includes, product description,
descriptive MoA, R&D brief, licensing and collaboration details & other developmental
activities, The report reviews key players involved Non-Hodgkin Lymphoma Market
therapeutics and enlists all their major and minor projects, The report assesses Non-Hodgkin
Lymphoma Industry therapeutics based on drug target, mechanism of action (MoA), route of
administration (RoA) and molecule type, The report summarizes all the dormant and
discontinued pipeline projects, The report reviews latest news related to pipeline therapeutics
for Neuropathic Pain.

Reason to Buy Non-Hodgkin Lymphoma Market Report-

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drove them from pipeline.
Raspberry-like poly(-glutamic acid) hydrogel
particles for pH-dependent cell membrane passage
and controlled cytosolic delivery of antitumor drugs
Authors Cho SH, Hong JH, Noh YW, Lee E, Lee CS, Lim YT
Received 21 July 2016
Accepted for publication 13 September 2016
Published 27 October 2016 Volume 2016:11 Pages 56215632
DOI https://doi.org/10.2147/IJN.S117862
Checked for plagiarism Yes
Review by Single-blind
Peer reviewers approved by Dr Akshita Wason
Editor who approved publication: Dr Thomas J Webster

Sun-Hee Cho,1,* Ji Hyeon

Hong,2,* Young-Woock Noh,1Eunji
Lee,2 Chang-Soo Lee,3 Yong Taik
Lim1

1SKKU Advanced Institute of

Nanotechnology, School of Chemical
Engineering, Sungkyunkwan
University, Suwon, Gyeonggi-
do, 2Graduate School of Analytical
Science and Technology, Chungnam
National University, 3Hazards Monitoring Bionano Research Center, Korea Research
Institute of Bioscience and Biotechnology, Yuseong-gu, Daejeon, Republic of Korea

*These authors contributed equally to this work

Abstract: In this research, we synthesized bioderived poly(amino acid) hydrogel particles

that showed pH-dependent membrane-disrupting properties and controlled cytosolic delivery
of antitumor drugs. Poly(-glutamic acid) (-PGA) that has been produced extensively using
bacteria, especially those of Bacillus subtilis species, was modified with cholesterol (-
PGA/Chol), and the -PGA/Chol conjugates were used to form polymeric nanoparticles the
size of 21.01.1 nm in aqueous solution. When the polymeric nanoparticles were mixed with
doxorubicin (Dox), raspberry-like hydrogel particles (RBHPs) were formed by the
electrostatic interaction between the cationically charged Dox and the anionically charged
nanoparticles. The average size and surface charge of the RBHPs in aqueous solution were
444.9122.5 nm and -56.44 mV, respectively. The loaded amount of Dox was approximately
63.9 g/mg of RBHPs. The RBHPs showed controlled drug release behavior in both in vitro
and ex vivo cell-based experiments. Through fluorescence microscopy and fluorescence-
activated cell sorting, the cellular uptake of RBHPs into human cervical cancer cells (HeLa)
was analyzed. The cytotoxic effect, evaluated by the methyl tetrazolium salt assay, was
dependent on both the concentration of RBHPs and the treatment time. The pH-dependent
membrane-disrupting properties of the RBHPs and the subsequent cytosolic delivery of Dox
were evaluated using a standard hemolysis assay. Upon an increase in hydrophobicity at the
lysosomal acidic pH, RBHPs could easily interact, penetrate cell membranes, and destabilize
them. Taken together, the data suggested that RBHPs could be used as drug delivery carriers
after loading with other therapeutic drugs, such as proteins or small interfering RNA for
cancer therapy.

OLIA Olive Oil

Substrate: Glass Bottle , Substrate: Wood, Category: Food

Jessica Deseo | June 10, 2014

Olive oil and gold, a combination that is becoming "the perfect taste of luxury." OLIA olive
oil is a premium extra virgin oil with organic and gold edible flakes. A beautiful package
design from Redfish Inspirations that combines natural wood with the extravagant look of gold
speckles. The outer rigid box combines a gold foil neck and black simplicity that houses this
olive oil perfectly.

"When liquid gold gets united with

real gold they do create the perfect
taste of luxury.
24K edible gold is a dietary habit
since ancient years. Eating gold
leaves appeared as a habit of the
great European families in the
beginning of the 19th century and
it was considered to be a synonym
of well-being and good life. Recent
medical studies prove the potent
antioxidant action of gold which
enhances the restructuring of cells
and the mental well-being. Gold is
transferred to the body through a
biodegradable water-soluble polymer, i.e. -PGA (gamma-Poly-Glutamic Acid) and by
developing properties such as the transfer of moisture to the cells, it configures immune
balance, by exercising strong anti-cancer action (Univ. of Kookmin/ Korea, 2005, a study
published by Department of Bio & Nano Chemistry).

Neuroplasticity, Neuroregeneration, and Brain Repair

Tuesday, June 13, 2017 - Wednesday, June 14, 2017

The New York Academy of Sciences, 7 World Trade Center, 250 Greenwich St Fl 40, New York,
USA
Astrocytes in Central Nervous System Repair and Regeneration
Michael V. Sofroniew, MD, PhD, David Geffen School of Medicine at the University of
California, Los Angeles, Los Angeles, California, United States

Astrocytes respond to all forms of central nervous system (CNS) damage. Molecular dissection in
vivo shows that astrocyte reactivity is a broad spectrum of potential changes ranging from mild
functional changes to overt scar formation. These changes are regulated by heterogeneous
molecular signaling mechanisms in a context specific manner. Genetic loss-of-function studies are
identifying diverse beneficial functions of reactive astrocytes, for example in regulating CNS
inflammation or synaptic reorganization after CNS insults. Other studies indicate that contrary to
long-standing dogma, newly-proliferated scarforming astrocytes aid, rather than inhibit, the
regeneration of damaged CNS axons. Roles for astrocytes in sustaining and restoring neural circuit
function after CNS injuries, including contributions to tissue repair, synaptic plasticity and
regeneration are being explored and identified. The potential for astrocyte dysfunction to
contribute to neural dysfunction is also being demonstrated. A conceptual framework is emerging
that encompasses both normal astrocyte reactivity that is critical for preservation of tissue and
function after CNS insults, and dysfunctional astrocyte reactivity which may be harmful through
loss-of-functions due to genetic defects, genetic polymorphisms, autoimmune attack, or chronic
exposure to inflammatory stimuli.

Anderson et al. (2016) Astrocyte scar formation aids central nervous system axon regeneration.
Nature 532:195-200
Sofroniew MV (2015) Astrocyte barriers to neurotoxic inflammation. Nat Rev Neurosci 16:249-
63
Burda JE, Sofroniew MV (2014) Reactive gliosis and the multicellular response to CNS damage
and disease. Neuron 81:229-248

Regulation of Synapses and Synaptic Strength

Richard Tsien, DPhil, New York University School of Medicine, New York, New York, United
States

Which brain disorders are truly diseases of the synapse full blown synaptopathies? If
dysfunctional synapses and abnormal excitability contribute, what are the other elements and how
do they all knit together? These questions recur in disorders ranging from Alzheimers to autism.
Here we address such generic issues in a specific case, by reporting our studies of autism spectrum
disorder (ASD), a monogenic, dominant form called Timothy Syndrome (TiS). Individuals with
TiS develop ASD with a >60% likelihood because of a point mutation in the pore-forming subunit
of a calcium channel (CACNA1C, encoding CaV1.2). In a mouse model (TS2-neo), we found
persistent and repetitive behavior, reduced social interaction, and altered vocalizations, impetus for
clarifying the pathophysiology. Dysfunctional homeostatic plasticity is often suggested as a
possible pathogenic mechanism for ASD. In studying homeostatic adaptation, we discovered
intriguing differences in the autoregulation of synaptic weights and intrinsic excitability in TS2-
neo cortical pyramidal cells subjected to action potential blockade by 24 hour tetrodotoxin (TTX)
treatment. TS2-neo pyramidal neurons exhibited an exaggerated upregulation in amplitude and
frequency of unitary events, accompanied by higher intensity of GluA1-containing AMPA
receptors. Homeostatic adaptation of intrinsic excitability was similarly exaggerated. We observed
similar electrophysiological alterations in layer 2/3 pyramidal neurons in primary visual cortex
recorded ex vivo from visually deprived WT and TS2 animals. These results fit with notion that
homeostatic autoregulatory effects can be abnormally large in ASD. They also provide novel
perspective on how CaV1.2 operates as an activity reporter.

Coauthors: Simon D. Sun, BS, Ben S. Suutari, BS, Boxing Li, PhD
New York University School of Medicine, New York, New York, United States

Striatal Plasticity in Parkinson's Disease

D. James Surmeier, PhD, Department of Physiology, Feinberg School of Medicine, Northwestern
University, Chicago, Illinois, United States
Parkinsons disease (PD) is the second most common neurodegenerative disease in the United
States. The core motor symptoms of PD are a consequence of the degeneration of dopaminergic
neurons that innervate the striatum. This innervation not only modulates the short-term excitability
of striatal neurons but also shapes the strength of corticostriatal synapses in a way that regulates
goal-directed and habitual actions. The loss of this innervation leads to an imbalance in the
excitability of striatal circuits modulating basal ganglia circuits, leading to the hypokinetic features
of the disease. It is widely appreciated that the striatal imbalance stems from the differential
expression of dopamine receptors by direct pathway spiny projection neurons (dSPNs) and
indirect pathway spiny projection neurons (iSPNs). What is less appreciated is the extent to which
intrinsic and synaptic homeostatic plasticity, particularly in iSPNs, mitigate the consequences of
declining dopaminergic signaling, but distort striatal connectivity with the cerebral cortex.
Symptomatic therapies that restore basal levels of dopamine in the striatum can reverse alterations
in intrinsic excitability but fail to correct distortions in synaptic strength. Moreover, with
fluctuations in striatal dopamine levels, aberrant synaptic plasticity leads to the motor
complications or dyskinesia. The presentation will summarize recent work in mouse models of PD
exploring these adaptations in the striatal circuitry and strategies for reducing their impact on
symptom severity.

Coauthors: Asami Tanimura, PhD, Steven M. Graves, PhD, and Weixing Shen, PhD, Department
of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, United
States

Novel Mechanisms of Immune-mediated Nervous System Regeneration

Roman J. Giger, PhD, University of Michigan School of Medicine, Ann Arbor, Michigan, United
States

Innate immunity can facilitate nervous system regeneration, yet the underlying cellular and
molecular mechanisms are not well understood. We found that intraocular injection of
lipopolysaccharide (LPS), a bacterial cell wall component, or the fungal cell wall extract zymosan
both lead to rapid and comparable intravitreal accumulation of blood-derived myeloid cells.
However, when combined with retro-orbital optic nerve crush injury, lengthy growth of severed
retinal ganglion cell (RGC) axons occurs only in zymosan-injected mice, and not in LPS-injected
mice. In mice deficient for the pattern recognition receptor dectin- 1 but not Toll-like receptor-2
(TLR2), zymosan-mediated RGC regeneration is greatly reduced. The combined loss of dectin-1
and TLR2 completely blocks the pro-regenerative effects of zymosan. In the retina, dectin-1 is
expressed by microglia and dendritic cells, but not by RGCs. Dectin-1 is also present on blood-
derived myeloid cells that accumulate in the vitreous. Intraocular injection of the dectin-1 ligand
curdlan [a particulate form of (1, 3)-glucan] promotes optic nerve regeneration comparable to
zymosan in WT mice, but not in dectin-1(-/-) mice. Particulate (1, 3)-glucan leads to increased
Erk1/2 MAP-kinase signaling and cAMP response element-binding protein (CREB) activation in
myeloid cells in vivo. Loss of the dectin-1 downstream effector caspase recruitment domain 9
(CARD9) blocks CREB activation and attenuates the axon-regenerative effects of (1, 3)-glucan.
Studies with dectin-1(-/-)/WT reciprocal bone marrow chimeric mice revealed a requirement for
dectin-1 in both retina-resident immune cells and bone marrow-derived cells for (1, 3)-glucan-
elicited optic nerve regeneration. Collectively, these studies identify a molecular framework of
how innate immunity enables repair of injured central nervous system neurons.

Subtype-specific Local Growth Cone Control over Circuit Development, Regeneration, and
Degeneration
Jeffrey D. Macklis, MD, DHST, Department of Stem Cell and Regenerative Biology, and Center
for Brain Science, Harvard University, Cambridge, Massachusetts, United States

The formation of circuits throughout the nervous system, and within and from the cerebral cortex
in particular in this presentation, relies heavily on molecular machinery localized at the tips of
growing axons in structures termed growth cones (GCs). Subsets of neurons transcriptomes and
proteomes localize to GCs to implement growth and guidance of nascent axons toward their
specific targets. Until now, these subcellular and likely subtypespecific RNA and protein networks
have not been experimentally accessible directly from the brain. Here, I will report subtype-
specific GC sorting and subcellular RNA-Proteome mapping as a generalizable approach to reveal
and investigate local molecular machinery that drives brain circuit development. Applying this
approach to the long-range axon projections of callosal projection neurons connecting the two
hemispheres of the cerebral cortex, and to corticothalamic projection neurons, we identify that
native GCs 1) possess remarkably deep and rich molecular constituents for local synthesis,
folding, and turnover of select protein classes, suggesting function as "mini-cellular" and
significantly ~autonomous units; 2) that each subtype contains both quite distinct, subtype-specific
molecular machinery plus shared molecular machinery; 3) that hundreds of proteins and hundreds
of RNAs (coding and not) are enriched orders of magnitude in GCs compared to their own parent
somata, indicating subcellular polarity and isolation of functions; 4) that targeting motifs direct
subtype-specific GC localization. As one example, we identify a hub molecule regulating cell
growth found specifically in axon GCs rather than cell bodies, and that mRNA classes distribute
within developing projection neurons based on their sensitivity for translation. Essentially this
entire class of transcripts map specifically to GCs and not to their parent cell bodies. Given the
importance of growth control for axon growth and regeneration, this subcellular organization
might also have implications for future regenerative strategies in the nervous system. Subtype-
specific GC sorting and differential subcellular RNA-proteome mapping is applicable to any
labeled neurons in the nervous system, enabling identification of specific molecular substrates of
circuit development, regeneration, and mis-wiring causing neuronal circuit pathology. The ability
to directly compare multiple distinct GC-soma subtypes using multi-color sorting makes this
approach broadly applicable to future studies in the fields of axon guidance, neurodevelopmental
disorders, and neuron reprogramming. Subcellular RNA-proteome mapping enables identification
of distinguishing molecular constituents of subtype-specific GCs with complex and unique
trajectories that ultimately target distinct brain areas. Further, it enables direct molecular
investigation of subtype-specific GCs compared with GCs from mutant, regenerative, non-
regenerative, or reprogrammed neurons to discover molecular mechanisms of circuit development,
mis-wiring, and regeneration.

Intermittent Bioenergetic Challenges Bolster Brain Resilience

Mark P. Mattson, PhD (1,2)
(1) Laboratory of Neurosciences, National Institute on Aging Intramural Research Program,
Baltimore, Maryland, United States
(2) Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore,
Maryland, United States

Evolutionary considerations suggest that the brain should function well/optimally during periods
of food deprivation/fasting and physical exertion. Brain-intrinsic pathways and peripheral signals
by which fasting and exercise promote synaptic plasticity, neurogenesis and resilience under
stressful conditions are being elucidated. Our findings suggest that running and fasting can
stimulate mitochondrial biogenesis in neurons by mechanisms involving BDNF signaling and
PGC-1, a pathway critical for the formation and maintenance of synapses. The mitochondrial
protein deacetylase SIRT3 mediates adaptive responses (stress resistance and modulation of
neuronal network activity) of neurons to exercise and fasting. By mechanisms involving
deacetylating SOD2 and cyclophilin D, SIRT3 protects neurons in animal models of acute brain
injury and neurodegenerative disorders. Interestingly, behavioral adaptations to intermittent fasting
(reduced anxiety and preservation of cognitive performance) involve SIRT3-dependent
enhancement of GABAergic tone in the hippocampus, which may also protect against
excitotoxicity. Regarding peripheral signals generated in response to fasting and vigorous exercise,
we found that the ketone -hydroxybutyrate, which is produced from fatty acids during fasting and
vigorous exercise, can stimulate BDNF production, which may mediate beneficial effects of these
bioenergetic challenges on neuronal plasticity and stress resistance. Additional data suggest that
combining energy restriction with exercise can elicit additive or synergistic beneficial effects on
neuroplasticity and performance. Collectively, the emerging picture of bioenergetics and brain
health reveals that intermittent energy restriction and exercise promote neuroplasticity and
resistance to injury and disease, whereas a couch potato lifestyle fosters suboptimal brain
function and poor resilience. Supported by the NIA Intramural Research Program.

Malfunctioning Autophagy and its Pathway to Neurodegeneration

Ana Maria Cuervo, MD, PhD, Albert Einstein College of Medicine, Bronx, New York, United
States

Autophagy is an essential cellular process that contributes to cellular quality control and to
maintenance of the cellular energetic balance. Failure of different types of autophagy in neurons
has been shown to result in major alterations in neuronal proteostasis, energetics and functioning
and have been linked to severe neurodegenerative disorders. We have identified a dynamic cross-
talk among different forms of autophagy that is used to protect cells from failure in any of these
systems and to avoid toxic effects of pathogenic proteins on them. To gain a better understanding
of the consequences of autophagy malfunctioning in neurons and on the rules that govern the
autophagic cross-talk we have developed a series of mouse models with systemic or tissue-specific
blockage of a selective form of autophagy known as chaperone-mediated autophagy (CMA).
Analysis of these models has revealed that added to the previously known role of CMA as part of
the cellular response to stress, this type of autophagy is also required in the regulation of important
cellular processes such as metabolism of lipids and carbohydrates, cell cycle, cell reprograming
and cellular differentiation. Phenotypic characterization of these mice is allowing us to link CMA
deficiency with different age-related neurodegenerative diseases.

Mitochondrial-linked Mechanisms and Therapeutic Opportunities

Valina L. Dawson, PhD (1,2)
(1) Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Departments of
Physiology, Neurology and the Solomon H. Snyder Department of Neuroscience, Johns Hopkins
University School of Medicine, Baltimore, Maryland, United States
(2) Adrienne Helis Malvin Medical Research Foundation, New Orleans, Louisiana, United States

Parkinson's disease (PD) is a common neurodegenerative disease of complex etiology with

roughly 90% of PD cases considered sporadic, and 10% attributable to familial mutations.
Mutations in the parkin gene, which encodes the E3 ligase parkin, are the most common cause of
autosomal recessive PD. To date, more than 100 pathogenic parkin mutations disrupt the protein's
E3 ligase activity, either directly or by altering the solubility or stability of the protein, leading to
dopaminergic cell death. Importantly, recent evidence from post-mortem PD brain samples and
mouse models suggest that parkin is inactivated by post-translational modifications, including
oxidation, nitrosylation, addition of dopamine and phosphorylation by c-Abl, an important stress-
activated non-receptor tyrosine kinase that is activated in sporadic PD brains and in animal models
of PD. Thus, inactivation of parkin maybe a common and critical feature of PD. PARIS (ZNF746)
is a pathologic parkin substrate, which contributes to DA neuronal loss in animal models of parkin
inactivation. PARIS accumulation plays a repressive function against the transcriptional
coactivator, peroxisome proliferator-activated receptor gamma coactivator-1-alpha (PGC-1)
expression, thought to be critical for DA neuron survival. Consistent with its role in DA neuronal
toxicity, PARIS accumulates in mouse brain with parkin inactivation and importantly in human
sporadic and familial PD brains. Dysfunction of parkin has profound effects on mitochondrial
quality control (autophagy, transport, fusion, fission and biogenesis), with PARIS playing an
important role in mediating parkins effect in biogenesis. Disruption of the homeostatic
mechanisms that controls the content of mitochondria in a cell through degradation (mitophagy)
and synthesis (biogenesis), can lead to neurodegeneration and significantly contribute to the
pathogenesis of PD.
Human Glial Progenitor Cell-based Treatment and Modeling of Neurological Disease
Steven A. Goldman, MD, PhD (1,2)
(1) University of Copenhagen Faculty of Medicine, Copenhagen, Denmark
(2) University of Rochester Medical Center, Rochester, New York, United States

The most abundant precursor cells of the adult human brain are glial progenitor cells (GPCs),
which can give rise to both astrocytes and oligodendrocytes. As a result, diseases of glia may
provide readily accessible targets for cell-based therapies. The glial diseases, which include the
myelin disorders as well as those referable to astrocytic dysfunction, are among the most prevalent
and disabling conditions in neurology, and may be particularly appropriate targets for progenitor
cell-based therapy. This talk will focus on the potential utility of transplanting pluripotent stem
cell-derived GPCs as a means of treating the diseases of myelin, as well as of those
neurodegenerative disorders with significant astroglial involvement. I will also discuss the utility
of the glial chimeric mice that result from the neonatal implantation of human GPCs into the
mouse brain. In these mice, the human glial progenitors out-compete their murine counterparts to
eventually dominate the glial population of the recipient brains. Human glial chimerization has
significant effects on neurophysiology and behavior, which suggest the importance of human-
specific glial attributes to neural network function. By generating human glial chimeric mice using
patient-derived hiPSC-derived GPCs, we may now investigate the causal contributions of glia to
human brain disease, by producing disease-specific human glial chimeras. These mice provide us
a new model system by which to study not only the myelin disorders, but the entire range of
neurodegenerative and neuropsychiatric diseases in which glia may causally participate.

Stem Cell-derived Astrocytes for the Treatment Neurodegenerative Diseases

Clive Svendsen, PhD, Cedars-Sinai Medical Center, Los Angeles, California, United States

Astrocytes and growth factors play a key role in brain health and functioning. We have developed
a reliable way of expanding human neural progenitors from both fetal tissue and iPSCs as
organoids without losing cell/cell contact. These organoids transition from making neurons to
astrocytes at around passage 20. They can also be genetically modified to release powerful growth
factors such as GDNF. We have been developing translational approaches to use these cells for
treating amyotrophic lateral sclerosis (ALS), parkinson's disease (PD), huntington's disease (HD),
alzheimers disease (AD), and retinal diseases. For ALS we have initiated a Phase 1/2a clinical
trial in 18 patients to establish whether this approach can slow degeneration and maintain function.

Development of Functionally Heterogeneous Astrocytes in Mammalian Central Nervous

System
David H Rowitch (1,2)

Astrocytes are heterogeneous in terms of morphology, gene expression and physiological

properties. How is such diversity determined? This talk will focus on advances in understanding
astrocyte development including the role of neural tube patterning in specification and
developmental functions of astrocytes during synaptogenesis, axon path finding and motor neuron
survival. We propose that a precise understanding of astrocyte development is critical to defining
heterogeneity and could lead to a better appreciation for their roles in support of local-regional
neural circuits. For example, we have shown that ventral spinal cord astrocytes express Sema3a,
which is essential for synaptogenesis and motor neuron survival. New screening approaches could
indicate diversification of cortical astrocytes conferring layer-specific functions. Such information
leads to the proposal that optimized cell-based neuronal therapies would be augmented by co-
transplant of regional specialized astrocytes.

Coauthors: Omer Bayrakartar (1,2), Anna V Molofsky (2), Kevin W. Kelley (2), Hui Hsin Tsai
(2), Huiliang Li (3), Raquel Taveira Marques (3), Helin Zhuang (3), Arturo Alvarez-Buylla (2),
Robert Krencick (2), Erik M Ullian (2), Nicola Allen (4), William D Richardson (3)
(1) University of Cambridge, Cambridge, United Kingdom
(2) University of California, San Francisco, San Francisco, California, United States
(3) University College London, London, United Kingdom
(4) Salk Institute, La Jolla, California, United States

Industry Perspective Lecture

Disease-modifying Drugs for Alzheimer's Disease The Past and The Future
Eric Karran, BSc, PhD, Foundational Neuroscience Center, AbbVie, Cambridge, Masschusetts,
United States
Alzheimers disease is a devastating neurodegenerative disease that follows an unremitting course
leading to severe cognitive impairment. Increasing age represents the greatest risk factor and as the
population shifts to an increasingly elderly demographic, the incidence and prevalence of this
disorder will likely increase. No therapies are available that slow or prevent the progression of the
disease, and recently, promising agents have failed in phase 3 clinical testing. I will review
previous clinical trials to determine what might be learned, and consider what future trials may
reveal.

Is Alzheimer's Disease Caused by Long-term Depression Gone Awry?

Graham Collingridge, FRS, FMedSci, FSB, FBPhS (1,2,3)
(1) Department of Physiology, University of Toronto, Toronto, Canada
(2) Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Canada
(3) School of Physiology, Pharmacology & Neuroscience, University of Bristol, Bristol, United
Kingdom

There is growing evidence that dysregulated synaptic plasticity is a contributing factor in a wide
variety of neurological and psychiatric disorders. We have proposed that one form of synaptic
plasticity, N-methyl-D-aspartate (NMDA) receptor-dependent long-term depression (LTD),
becomes overactive and triggers synapse elimination and the associated cognitive deficits in
neurodegenerative conditions, such as Alzheimers disease. NMDAR-LTD is particularly
prevalent early in development where it is likely to be involved in the physiological pruning of
superfluous synaptic connections. It is then largely down regulated. We propose that a variety of
genetic and environmental factors may lead to the inappropriate reactivation of NMDAR-LTD and
that this constitutes an early and causal element in neurodegenerative conditions. In support of this
hypothesis, we have found that a variety of molecules that are closely associated with Alzheimer's
disease are integral components of the LTD process. Such a mechanism can explain the link
between Abeta and tau and can, in theory, underlie both Abeta-dependent and Abeta-independent
forms of neurodegenerative disorders.
Modeling Human Brain Development and Disorders using Human Induced Pluripotent
Stem Cells (hiPSCs)
Guo-li Ming, MD, PhD, Department of Neuroscience, University of Pennsylvania, Philadelphia,
Pennsylvania

Three dimensional (3D) cerebral organoid cultures from human iPSCs have been recently
developed to recapitulate the cytoarchitecture of the developing brain. This system offers unique
advantages in understanding molecular and cellular mechanisms governing embryonic neural
development and in modeling congenital neurodevelopmental disorders, such as microcephaly.
We have improved the organoid technology and developed a robust protocol to produce forebrain-
specific organoids derived from human iPSCs using a novel miniaturized spinning bioreactor that
recapitulate the human embryonic cortical development. ZIKV, a mosquito-borne flavivirus, has
re-emerged as a major public health concern globally because ZIKV causes congenital defects,
including microcephaly, and is also associated with Guillain-Barr syndrome in infected adults.
We found that ZIKV exhibit specific tropism towards human neural progenitor cells and results in
cell death and defects in neural development. I will discuss our recent work in further dissecting
the molecular mechanisms underlying the ZIKV pathogenesis and microcephaly.

Engineering of Neurogenesis via Lineage Reprogramming

Benedikt Berninger, PhD (1)

Engineering new neurons via lineage reprogramming provides new avenues for brain repair. We
have previously found that reprogramming of glia into induced neurons in the adult mouse
cerebral cortex was dependent on prior injury. One of the hallmarks of the injury response is
increased glial proliferation. We reasoned that if proliferation is a property conducive for lineage
conversion that glial populations that undergo expansive cell division during postnatal brain
development may be endowed with a greater reprogramming competence. Indeed we found that
forced expression of Ascl1 and Sox2 (AS) or Neurog2 and Bcl2 (NB) resulted in the generation of
neurons in the absence of a prior injury. Interestingly NB- and AS-induced neurons differed
markedly in their physiological properties, with the former developing in more mature neurons in
terms of morphology and physiology. Choice of reprogramming factors is likely to impact the
conversion trajectory. Using single cell RNA-sequencing we found that AS-induced
reprogramming of adult human brain pericytes into induced GABA-positive neurons is not
accomplished via immediate replacement of the pericyte transcriptional program by a terminal
neuronal program, but involves the transition through a interneuron precursor-like state, albeit in
the absence of cell division. Intriguingly, successful induction of the precursor state depends on the
synergism between the two transcription factors as either factor alone results in the induction of
gene expression profiles that markedly diverge from that induced when expressed conjointly. Our
data reveal part of the molecular logic that drives lineage conversion of non-neural cells towards a
GABA neuron identity.

Coauthors: Marisa Karow, PhD (1,2), Sophie Pron, PhD (1), Nicols Marichal, PhD (1), Sven
Falk, PhD (2), Gray Camp, PhD (3), Barbara Treutlein, PhD (3)
(1) University Medical Center Mainz of the Johannes Gutenberg University Mainz, Mainz,
Germany
(2) Biomedical Center Munich, Ludwig-Maximilians University of Munich, Munich, Germany
(3) Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany

Rejuvenating and Re-engineering Aging Memory Circuits

Amar Sahay, PhD (1,2,3,4)
(1) Center for Regenerative Medicine, Massachusetts General Hospital, Boston, Massachusetts,
United States
(2) Harvard Stem Cell Institute, Cambridge, Massachusetts, United States
(3) Department of Psychiatry, Massachusetts General Hospital, Harvard Medical School, Boston,
Massachusetts, United States
(4) BROAD Institute of Harvard and MIT, Cambridge, Massachusetts, United States

Episodic memory impairments and loss of memory precision are hallmarks of age-related
cognitive decline and mild cognitive impairment (MCI). Studies by us and others have implicated
adult hippocampal neurogenesis in resolution of memory interference through population based
coding mechanisms that support pattern separation. Here, I will present two complimentary
approaches, modulation of neuronal competition dynamics and molecular control of granule cell
recruitment of inhibition, by which we can rejuvenate and re-engineer the dentate gyrus-CA3
circuit to improve memory precision in adulthood and aging.
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