**5.2. Thresholding and motor drive**

The control of robotic prosthesis is provided through the thresholding technique [21]. Once the signal is received in digital format, taking all necessary considerations as described before, a suitable threshold is applied to that particular quantized digital signal.

Before applying the threshold, the digital quantized signal is to be observed properly. A threshold value should then set be accordingly. It is recommended to set the threshold value to a point which is less than half the digital quantized output of the EMG signal. When the digital signal exceeds this threshold, the microcontroller should set an output pin to '1' and '0' otherwise [21]. E.g. if the maximum value of the digital quantized signal is 750 (decimal value) then we can set a threshold of 275. This signal is forwarded to an H-bridge or a motor driver in order to drive the respective electric motors of a robotic mechanism.

The motor driver should be designed or selected according to our requirements of electric motor. Usually a motor driver which can drive a 12V motor and handle up to 4A current can adequately meet requirements for a robotic arm.


**Abstract**

Polyesteramides PNOBDME (C34H38N2O6)n, Poly[oxy(1,2-dodecane)-oxycarbonyl-1,4-phenylene-amine-carbonyl-1,4-phenylene-carbonyl-amine-1,4 phenylene-carbonyl], and PNOBEE (C26H22N2O6)n, Poly[oxy(1,2-butylene) oxy-carbonyl-1,4-phenylene-amine-carbonyl-1,4-phenylene-carbonyl-amine-1,4-phenylene-carbonyl], have been designed and synthesized as cholesteric liquid crystals (LCs)—through a condensation reaction between 4- 4′-(terephthaloyldiaminedibenzoic chloride) (NOBC) and racemic glycol, DL-1,2-dodecanediol or DL-1,2-butanediol, respectively—as chemical modifications of multifunctional cholesteric LC polyesters, involving new properties but holding the precursor helical macromolecular structures. The new compounds have been characterized by 1 H and 13C-NMR, COSY and HSQC, exhibiting two 1 H-independent sets of signals observed for each enantiomer, attributed to two diastereomeric conformers, *gg* and *gt*, of the torsion containing the asymmetric carbon atom in the spacer. They have also been characterized by x-ray diffraction with synchrotron radiation source. Thermal behaviour of the new compounds is studied by thermogravimetric (TG) and differential scanning calorimetry (DSC) analysis. The substitution of the ester groups in the mesogen by amide groups causes an increase of thermal stability with respect to the precursors. Optical rotatory dispersion (ORD) is evaluated. Morphology of powdered PNOBDME exhibits spherical clusters of about 5 μm in diameter homogeneously dispersed. Molecular models show helical polymeric chains with stereoregular head-tail, isotactic structure, explained as due to the higher reactivity of the primary hydroxyl with respect to the secondary one in the glycol through the polycondensation reaction. Besides being biocompatible, these synthetic polyesteramides have proved to act as non-viral vectors in gene therapy and be able to transfect DNA to the nucleus cell. Similar new cationic cholesteric liquid crystal polyesters have also been synthesized in our laboratory.

**Keywords:** cholesteric LC polymer, biocompatible polyesteramides, synthesis, characterization, SAXS/WAXS, molecular simulation

## **1. Introduction**

In 1944, Erwin Schrödinger, one of the founders of quantum mechanics, completed in Dublin a small book entitled *What Is Life? The Physical Aspect of the Living Cell* [1]. He predicted there the concept of "aperiodic crystal", to define a gene or perhaps the whole fibre of the chromosome, "that in my opinion, is the material

carrier of life…," "able to grow in aggregates bigger and bigger without the clumsy resource of repetition in three directions" as crystals do [2].

The concept of "aperiodic crystal" was surprisingly premonitory 9 years before the structure of DNA fibres was determined by x-ray diffraction as a helical structure by Franklin [3] ("Photograph 51" in **Figure 1(a)**), Wilkins [5] and Watson and Crick who reported the double helix with periodic order along the sugar-phosphate helical backbone, every 34 Å, and with the *aleatory* distribution of the complementary base pairs [4] (**Figure 1(b)**). The sequence of base pairs along the structure, without lateral periodic order, being convenient to endow DNA of their capacity to store genetic information.

In 1988 Ringsdorf evidenced the parallelism between thermotropic and lyotropic liquid crystals (LCs) in materials science and lipids in life science, with common amphiphilic nature [6]. Both self-organize their molecules in supramolecular systems, leading to functional units in highly oriented systems, exhibiting new properties. The importance of lyotropic LCs for the life sciences has been known for a long time as a prerequisite for the development of life and the ability of cells to function. In materials sciences, the concept of function through organization led to the development of new liquid crystalline materials for advanced applications.

Since 1992 the International Union of Crystallography redefined the concept of crystal as: "Any solid which has a diffraction pattern essentially discrete" (Fourier space) [7]. Since then, the *crystal family* was accepted to be composed of *periodic* and *aperiodic crystals*, and liquid crystals belong to the last group.

With increasing temperature, they do not directly go from the crystalline state into the melt, but, in the middle, they undergo a *mesophase* state which combines the order of perfect crystals and mobility of liquids.

All mesophases exhibit long-range orientational order, by keeping the parallel orientation of their longitudinal molecular axes. Two major classes can be

#### **Figure 1.**

*(a) Photograph 51 by [3]; (b) DNA crystal structure according to [4]. Details of double-stranded PolyA-PolyT and PolyC-PolyG are shown at the right-hand side.*

**81**

**Figure 3.**

**Figure 2.**

*Cholesteric Liquid Crystal Polyesteramides: Non-Viral Vectors*

**1.1 Synthetic cholesteric liquid crystal polymers**

DL-1,2-dodecanediol or DL-1,2-butanediol, respectively.

differentiated: *nematic* (with molecular centres distributed isotropically) and *smectic* (with their molecular centres organized in layers). The special array of nematic planes stacked in a helical superstructure with a prevalent screw direction is called

DNA behaves as liquid crystal with the special array of the nematic planes, containing complementary base pairs, stacked in a superstructure with chiral helical symmetry of the charge distribution [8–10]. **Figure 3** shows micrographs of the

Two multifunctional cholesteric liquid crystal polyesters (ChLCP), named PTOBDME [C34H36O8]n and PTOBEE [C26H20O8]n, with chemical formulations in **Figure 4**, were synthesized in our laboratory—through a condensation reaction between 4-4′-(terephthaloyl-dioxybenzoylchloride) (TOBC) and racemic glycol,

Although only racemic materials were used in their synthesis, a cholesteric, chiral morphology, theoretically unexpected, was found for PTOBDME, m = 9 in **Figure 4**. Evidence of this was obtained when a white solid, recrystallized as the second fraction from toluene mother liquor after the filtration of the polymer, was

result had been previously attained for liquid crystal PTOBEE, m = 1 in **Figure 4**.

(0.0056 mol/l, toluene). The synthetic method [11, 12], based on the previously reported by Bilibin [13, 14], leads to the obtaining of two or more fractions with progressively enriched diastereomeric excess. Its structure and diastereomeric

The structure of these optically active cholesteric LC polymers was characterized by NMR, Raman spectroscopy, steady-state fluorescence and SAXS/WAXS [16, 17], as rigid or semirigid helical LC polymers chains with flexible branches (chiral groups are located in the backbone), **Figure 5**, one of the major types of

Both polymers behave as thermotropic and lyotropic. They self-assemble in nanocavities in solution, with different conformations depending on the solvent

*Types of liquid crystal mesophases: (a) nematic, (b) smectic a and (c) cholesteric, helical pitch.*

*Cholesteric mesophase of liquid crystal DNA exhibiting columnar hexagonal phase.*

589 = −1.43 (1.538 g/100 ml, toluene). A similar

25

589 = −2.33

25

Its second fraction was isolated as –PTOBEE, with a value of [α]

macromolecules forming the cholesteric mesophase [18].

cholesteric mesophase of DNA as the columnar hexagonal phase.

*DOI: http://dx.doi.org/10.5772/intechopen.91317*

the cholesteric mesophase (**Figure 2**).

identified as –PTOBDME, with [α]

excess could be characterized by NMR [15].

*Cholesteric Liquid Crystal Polyesteramides: Non-Viral Vectors DOI: http://dx.doi.org/10.5772/intechopen.91317*

*Liquid Crystals and Display Technology*

store genetic information.

carrier of life…," "able to grow in aggregates bigger and bigger without the clumsy

In 1988 Ringsdorf evidenced the parallelism between thermotropic and lyotropic liquid crystals (LCs) in materials science and lipids in life science, with common amphiphilic nature [6]. Both self-organize their molecules in supramolecular systems, leading to functional units in highly oriented systems, exhibiting new properties. The importance of lyotropic LCs for the life sciences has been known for a long time as a prerequisite for the development of life and the ability of cells to function. In materials sciences, the concept of function through organization led to the development of new liquid crystalline materials for advanced applications.

Since 1992 the International Union of Crystallography redefined the concept of crystal as: "Any solid which has a diffraction pattern essentially discrete" (Fourier space) [7]. Since then, the *crystal family* was accepted to be composed of *periodic*

With increasing temperature, they do not directly go from the crystalline state into the melt, but, in the middle, they undergo a *mesophase* state which combines

All mesophases exhibit long-range orientational order, by keeping the parallel orientation of their longitudinal molecular axes. Two major classes can be

*(a) Photograph 51 by [3]; (b) DNA crystal structure according to [4]. Details of double-stranded PolyA-PolyT*

and *aperiodic crystals*, and liquid crystals belong to the last group.

the order of perfect crystals and mobility of liquids.

The concept of "aperiodic crystal" was surprisingly premonitory 9 years before the structure of DNA fibres was determined by x-ray diffraction as a helical structure by Franklin [3] ("Photograph 51" in **Figure 1(a)**), Wilkins [5] and Watson and Crick who reported the double helix with periodic order along the sugar-phosphate helical backbone, every 34 Å, and with the *aleatory* distribution of the complementary base pairs [4] (**Figure 1(b)**). The sequence of base pairs along the structure, without lateral periodic order, being convenient to endow DNA of their capacity to

resource of repetition in three directions" as crystals do [2].

**80**

**Figure 1.**

*and PolyC-PolyG are shown at the right-hand side.*

differentiated: *nematic* (with molecular centres distributed isotropically) and *smectic* (with their molecular centres organized in layers). The special array of nematic planes stacked in a helical superstructure with a prevalent screw direction is called the cholesteric mesophase (**Figure 2**).

DNA behaves as liquid crystal with the special array of the nematic planes, containing complementary base pairs, stacked in a superstructure with chiral helical symmetry of the charge distribution [8–10]. **Figure 3** shows micrographs of the cholesteric mesophase of DNA as the columnar hexagonal phase.


**7. Nidus Vespae**

Nidus Vespae is widely distributed in China and is typically harvested in the autumn & winter seasons and dried in the open air, after removal of dead wasps, for use in traditional Chinese medicine, where it has been used in the treatment of a variety of diseases, including cardiovascular, digestive and urinary disorders. The well-known pharmacopoeia of traditional Chinese medicine also lists the use of Nidus vespae For toothaches, through tooth brushing. A study showed significant inhabitation of glucosyltransferases activity and biofilm formation by Nidus Vespa extract. The researchers concluded it to be a promising natural product for the prevention of dental caries. Nidus Vespa have been extensively used in traditional Chinese medicine, given their multiple pharmacological activities, including antimicrobial, anti-inflammatory, anti-virus and anesthetic properties (Xiao et al 2007).


**4. Iron uptake systems of uropathogenic** *Escherichia coli*

Urinary tract has limited iron. However, UPEC are able to produce small iron chelator molecules, known as siderophores, to scavenge ferric iron (Fe3+) in the host. The most prominent ones are yersiniabactin, salmochelin, and aerobactin [48, 49]. The yersiniabactin and its receptor, FyuA, are encoded in a PAI [50, 51]. It has also been reported that for efficient biofilm formation by UPEC, FyuA is required [52]. UPEC also secretes another important hydroxamate siderophore called aerobactin. This is produced from the condensation of two lysine and a citrate molecules. During UPEC invasion, the bacterium secretes salmochelin. Its outer membrane siderophore receptor (IroN) transports different catechol siderophores, including N-(2,3-dihydroxybenzoy)-L-serine and enterochelin also called enterobactin [53]. Enterobactin has less solubility and stability than

*Virulence Factors of Uropathogenic* Escherichia coli *DOI: http://dx.doi.org/10.5772/intechopen.99891*

#### **Figure 1.**

*UPEC-associated fitness and virulence. Adapted from the work by Servin [64].*

aerobactin [54–56] but has higher iron affinity than aerobactin in aqueous [55, 57]. UPEC also uses enterobactin for Fe3+ scavenging in the urinary tract [9]. However, enterobactin can be inactivated by the host proteins such as serum albumin and siderocalin thereby preventing its uptake [58]. UPEC overcomes this instability by modifying the enterobactin to salmochelin by glucosylation through the enzymatic action of glucosyltransferase and prevents it from being recognized by the host proteins [9]. Also, UPEC has another iron acquisition system called haemin uptake system consisting of Ton-B dependent receptor (ChuA) and heavy metal associated (*Hma*) receptor that takes part in direct upregulation of haem receptors from free iron during UPEC infection. This system has also been reported to play significant role in the formation of biofilm [59–61]. The expression of ChuA is controlled by other regulatory proteins. It has been reported that the production of ChuA is triggered as RfaH increases [62]. However, Hma does not depend on ChuA and it is controlled by Tyr-126. Both Hma and ChuA are associated with haem uptake for optimal kidney utilization [63]. **Figure 1** shows the diagram of UPEC-associated fitness and virulence factors.


**6. Storage and transport**

*Oral and Maxillofacial Surgery*

the lymph node itself [5].

**5.1 Saliva**

sis of head and neck lesions.

*Saliva is useful for testing:*

b.For caries activity tests

**5.2 Collection of specimens**

collection.

for at least 1 day.

5.Collect saliva for 10 min [6].

a.As an index to metabolic processes

c.For detection of various metabolites in smokers

FNAC. FNAC of cervical lymph nodes is a well-accepted diagnostic test of choice in both adult and pediatric patients for reliably distinguishing between benign/ reactive and malignant processes and guiding patient management with simple observation or antimicrobial therapy for infections, chemotherapy and radiation therapy or the need for more sample tissue like core biopsies or excisional biopsy of

Thus FNAC is recommended as a safe, quick and inexpensive tool in the diagno-

Collection of human saliva offers a noninvasive method for monitoring the disposition of unbound (free) drugs and many endogenous biomarkers. Human genomic DNA extracted from buccal epithelial cells and white blood cells found in saliva can be used in various applications in diagnostics. The correlations between blood and saliva biomolecule/biomarker concentrations range from good to excellent. Methods of collecting saliva range from simply spitting into a collection cup or using absorbent pads or swabs or the trademarked collecting devices. Freeze-thaw techniques are often employed to help break up the mucin protein that is responsible for the sticky, foamy saliva. There are few inherent drawbacks for using saliva as an ideal biofluid. In some cases, the drug, metabolites or other compounds being assayed may bind to absorbent materials, thus reducing recovery or giving a misleading result [6].

Whole saliva is commonly collected by draining, spitting, suction, and swab or absorbent method. Common stimuli used are chewing on paraffin wax and chewing gum at a fixed rate. As described by Wong D, the proposal for standardized collection of whole and glandular saliva can be followed for saliva sample collection.

1.Collect saliva samples at the same time of the day between 9:00 and 11:00 a.m.

2.Patient should refrain from eating and drinking at least 90 min before advised

3.If present, drug usage should be stopped that might affect salivary secretion

4.Rinse mouth with preferably deionized water prior to the saliva collection.

Collection of saliva into ice-cooled vials is recommended to slow down the activity of hydrolytic enzymes present in saliva in air-cooled preset environment. Proprietary collection vials contain a cocktail of protease inhibitors and

**52**

After collection, salivary samples must be snapped frozen in liquid nitrogen. In the absence of liquid nitrogen, freezing in dry ice is a practical choice when samples are collected. For a prolonged storage, −80°C temperature is preferred over storage at−20°C. The salivary samples can be diluted with glycerol (1:1) before storage. For immunochemical analysis such as ELISA, the saliva can be stored frozen after dilution (e.g., 1:100) in the assay buffer, usually PBS—0.5% Tween-20. To maintain the integrity of the proteins, before testing, the deep-frozen samples must be thawed as quickly as possible. Analysis of pH (H+ and HCO4 <sup>−</sup>) and viscoelastic properties is best analyzed in fresh saliva samples. Storage of salivary DNA and RNA is similar to that of a salivary protein sample [6].


**1. Introduction**

172 Gene Duplication

Vision, T.J., Brown, D.G., & Tanksley, S.D. (2000) The origins of genomic duplications in

Wallrapp, C., Verrier, S.B., Zhouravleva, G., Philippe, H., Philippe, M., Gress, T.M., & Jean-

Wang, Y., Chai, B., Wang, W., & Liang, A. (2010) Functional characterization of polypeptide release factor 1b in the ciliate Euplotes. *Biosci.Rep.*, Vol.30, pp. 425-431. Wang, Y. & Gu, X. (2000) Evolutionary patterns of gene families generated in the early stage

Wong, S., Butler, G., & Wolfe, K.H. (2002) Gene order evolution and paleopolyploidy in hemiascomycete yeasts. *Proc.Natl.Acad.Sci.U.S.A*., Vol.99, pp. 9272-9277. Zhouravleva, G., Schepachev, V., Petrova, A., Tarasov, O., & Inge-Vechtomov, S. (2006)

retrotransposition of GSPT1's mRNA? *IUBMB.Life*., Vol.58, pp. 199-202.

but does not carry eRF3-like activity. *FEBS Lett.*, Vol.440, pp. 387-392. Wang, W., Czaplinski, K., Rao, Y., & Peltz, S.W. (2001) The role of Upf proteins in

Wolfe, K. (2000) Robustness-it's not where you think it is. *Nat.Genet*., Vol.25, pp. 3-4.

Jean, O. (1998) The product of the mammalian orthologue of the *Saccharomyces cerevisiae HBS1* gene is phylogenetically related to eukaryotic release factor 3 (eRF3)

modulating the translation read-through of nonsense-containing transcripts. *EMBO*

Evolution of translation termination factor eRF3: Is GSPT2 generated by

Arabidopsis. *Science*, Vol.290, pp. 2114-2117.

of vertebrates. *J.Mol.Evol.*, Vol.51, pp. 88-96.

*J.*, Vol.20, pp. 880-890.

Though considerable sequence information from different organisms was available prior to the recent advances in genome sequencing technology, the foundation for our current understanding of the mechanisms of bacterial pathogenesis was laid by the release of the first complete genome sequence of *Heamophilus influenza* in 1995 (Fraser-Liggett, 2005). Ever since, significant progress in the availability of data for different genomes has been possible due to the contribution of various genome sequencing projects (Koonin & Wolf, 2008). Despite the complete genome sequences of many pathogenic organisms being available, the mortality rates due to these infectious agents still remains a problem, highlighting the need to decipher the complex molecular mechanisms responsible for survival of the bacteria. The wealth of complete genome information for pathogens can be effectively explored using comparative genomic tools for the identification of common and unique sets of genes involved in the propagation of virulence. Sequence comparison tools have been developed to identify homologous genes from the complete genomes of microorganisms. Homologous genes which arise from speciation tend to maintain functions similar to that of their ancestral molecule and are known as orthologs, while the genes originating from duplication events often evolve new functions and are defined as paralogs (Tatusov *et al.,* 1997).

The world of microbes is highly diverse with genome complexity differing across a wide range of microorganisms. In general, the difference in the complexity of genomes is dictated by the life style and environment of the organism (Cordero & Hogeweg, 2009). Life style plays an important role in regulating the genome dynamics of an organism, and functional novelty provided by gene duplication is thought to enhance the adaptation capability of the organism. In addition, horizontal transfer of operons or functional units of genes from external sources may provide an immediate functional benefit to the organism, thereby adding to the functional complexity of the genomes. The availability of complete genome sequences of important mycobacteria such as *Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycobacterium bovis, Mycobacterium leprae, Mycobacterium paratuberculosis, Mycobacterium avium* and others, can be used to gain deeper insights into possible

Analysis of Duplicate Gene Families

**2.2 Evolutionary analysis**

**3. Results and discussion**

**size**

BlastClust in two separate clustering procedures:

in Microbial Genomes and Application to the Study of Gene Duplication in *M. tuberculosis* 175

signature data from InterPro enabled the identification of approximately 27,827 proteins which exhibited complete domain identity (same InterPro matches) over their entire length to one or more proteins in *M. tuberculosis* strain H37Rv. Within each organism and across all organisms, the proteins showing complete domain identity were grouped together as duplicate gene sets or ortholog and paralog sets, respectively, and those with no common signature matches were considered to be single copies. In addition to the identification of expanded families using InterPro data, homologous sequences were clustered using

a. **Independent Genome Clustering**: This involves within genome clustering to generate clusters of paralogs or protein families for each genome. BlastClust was executed at a wide range of percentage identities over varying lengths of the sequence to select the optimum parameters. Amongst the tested parameters, a 30% similarity over 60% sequence length cut-off was chosen, as it generated a suitable number of clusters (in line

For evolutionary studies, in addition to 66 paralogous gene clusters, 116 multiple genome clusters from the phylogenetic matrix of six of the closely related mycobacterial genomes that showed gene family expansions in both *M. tuberculosis* and *M. leprae*, as well as other mycobacteria, were selected. The proteins in each of the clusters were aligned with T-coffee (Notredame *et al.*, 2000), and poorly aligned regions were edited using the Gblocks program (Castresana**,** 2000) with adjustments in the default settings for the generation of optimal sequence alignments. For each of these protein alignments, selection of the best-fitting amino acid substitution model was performed according to the Akaike informational criterion, and the gamma correction factor (alpha), the proportion of invariable sites (I), and observed amino acid frequencies (F) were estimated and selected for subsequent phylogenetic analysis using ProtTest (Abascal *et al.*, 2005). Since, PhyML is a maximum likelihood method with the ability to incorporate the estimated values of alpha, proportion of invariable sites, and observed frequencies, the tree topologies for the gene sets in the identified clusters were constructed using this program (Guindon & Gascuel, 2003). The genetic distance measures from each of the estimated tree topologies were used to compute

**3.1 Identification of expanded gene families and relation to GC content and genome**

We used sequence clustering and protein signature data to identify expanded genes families within and across several different microbial genomes. The across-genome clustering of protein sequence data yielded 1,984 expanded genes in 441 clusters for *M. tuberculosis*  H37Rv. The protein signature method allowed us to group 30,885 proteins into 2238 clusters from all the organisms. InterPro signatures usually match between 50% and 80% of a genome, so data is not available for every protein. Since signature data enables identification

with previously reported numbers of duplicated families for *M. tuberculosis*). b. **Multiple Genome Clustering**: In this, all of the 76 genomes were appended together for the clustering of related proteins (orthologs and paralogs). In addition, the clustering of six of the mycobacterial species was performed separately for the

evolutionary analysis of expanded gene families in *M. tuberculosis*.

average and maximum genetic distance using Perl scripts.

mechanisms of prokaryotic gene innovation. Genome data has been used in recent studies to compare different species of mycobacteria, as well as different strains, to understand the evolution and pathogenesis of *M. tuberculosis* (Marri *et al.,* 2006). In our study, duplicate gene sets from different mycobacteria were investigated to identify the distribution of important functional classes of protein families, and the evolution of these functional classes was further analyzed by comparing their genetic divergence following duplication.

The importance of gene duplication in prokaryotic gene innovation is well established and comparative analysis of duplicate genes with basic characteristic features of genomes like GC content and genome size may aid in deciphering their contributions. In contrast to eukaryotes, GC content varies widely across different bacterial genomes (Mann & Chen, 2010), and analysis of GC variations between related bacteria could be useful in establishing evolutionary relationships (Mann *et al.,* 2010). The focus of the majority of earlier studies was on deciphering the role of GC composition in HGT (Nelson *et al.,* 1999; Hamady *et al.*, 2006), transcription start and stop sites (Zhang *et al.,* 2004), nucleotide substitution rates (DeRose-Wilson & Gaut, 2007), optimal growth temperature (Basak & Ghosh, 2005; Musto, 2006) and metabolic characteristics (Naya *et al.,* 2002). Furthermore, genome size has been reported to increase with an increase in number of genes in duplicate gene families (Snel *et al.,* 2002; Pushker *et al.,* 2004). In this study we analyzed the genomes of 56 pathogenic and 20 non-pathogenic microorganisms to identify and characterize the expanded gene families across these organisms. In addition to the GC content, we investigated the relationship between genome size and duplicate gene percentage. On finding sufficient evidence for a correlation between genome size and extent of gene duplication, we further investigated the significance of duplicate genes in enhancing genome complexity. He and Zhang (2005) previously reported the importance of gene duplication in enhancing genome and organism complexity in eukaryotes. However, due to the difference in the selective pressures operating on prokaryotic and eukaryotic genomes, we used the duplicate and single copy genes to investigate the influence of protein lengths on genome and organism complexity of prokaryotic organisms, with a specific focus on investigating the role of duplicate genes in enhancing the genome complexity of *M. tuberculosis*.


*4.3. Model Comparison*

ST-CORAbico was compared with the Distribution Transformation method (DT) and the Gamma Quantile Mapping (GQM) method. Using the short- and long-lived storm scenarios that are presented above, Figure 9 presents the spatial differences and linear correlation between the total observed storms and the bias-corrected events obtained by ST-CORAbico, DT, and GQM. The results for both storm event scenarios showed that ST-CORAbico had the lowest spatial difference among the evaluated methods. For the short-lived storm scenario, ST-CORAbico displayed the highest correlation coefficient (*r*: 0.41) and the lowest RMSE and bias (RMSE: 4.05 mm; bias: 0.74) when compared with DT (*r*: 0.40; RMSE: 5.4 mm; bias: 1.17); and, GQM (*r*: 0.39 RMSE: 6.09mm and bias: 1.5). In the case of the long-lived storm, ST-CORAbico and DT showed a notable error reduction in contrast to the GQM method that showed the biggest differences. For this storm scenario, ST-CORAbico had the best performance (*r*: 0.71 RMSE: 18.02 mm; bias: 0.09), followed by DT (*r*: 0.68, RMSE: 23.0 mm; bias: 0.32), and finally GQM (*r*: 0.62, RMSE: 43.77 mm; bias: 0.97).

**Figure 8.** Satellite and bias corrected error distribution for short and long-lived events during monsoon seasons 2014–2017: (**a**,**b**) RMSE; (**c**,**d**) bias; and, (**e**,**f**) correlation coefficient.

**Figure 9.** Comparison between ST-CORAbico vs Distribution Transformation (DT) and Gamma Quantile Mapping (GQM).

Figure 10 presents the comparison between ST-CORAbico, DT, and GQM for short- and long-lived storm events. The boxplots show the distributions of the RMSE (Figure 10a,b), the bias (Figure 10c,d), and the correlation coefficient (Figure 10d,f) between the 25% and 75% percentiles for the original GPM-IMERG and the different bias correction methods. The dots represent the individual error for each storm event. In comparison with the two probabilistic methods, we found that ST-CORAbico consistently had the lowest RMSE as well as the lowest bias for both short- and long-lived storm events. ST-CORAbico and DT had a lower impact on the correlation coefficient, especially for short-lived events.

**Figure 10.** Comparison between the satellite GPM-IMERG (red), ST-CORAbico (blue), Distribution Transformation (green) and Gamma Quantile Mapping (grey) error dispersion during monsoon seasons 2014–2017. (**a**,**b**) RMSE; (**c**,**d**) bias; and, (**e**,**f**) correlation coefficient.


**1. Introduction**

Internationally, construction workers have higher rates of musculoskeletal disorders, and chronic diseases related to obesity, lack of physical activity and smoking than workers in other industries [1–4]. In construction workers, musculoskeletal disorders have a one-year pain prevalence rate (at least one

episode of pain in the last year) of 51% for the back, 37% for the lower extremities, 32% for the upper extremities, and 24% for the neck in construction workers [5]. The high prevalence of musculoskeletal and cardiovascular disorders causes a sizable burden to employers, insurers, and society as a whole, attributing to work absenteeism, healthcare costs, work schedule delays, and high turnover [6–8]. In 2014, approximately 33% of absenteeism was attributed to musculoskeletal symptoms [9].

Construction workers also have high rates of chronic health issues. Over 70% of construction workers are overweight [10]. Obese construction workers are at increased risk of receiving disability benefits for cardiovascular disease and musculoskeletal disorders [11,12]. This risk is even higher for obese workers with high physical job demands, especially for those with musculoskeletal disorders [11]. Specifically, construction workers also have the highest prevalence of smoking (39%) of all occupational groups [1]. The risk of chronic lung disease and cancers is also amplified by the combined effects of smoking with other respiratory exposures, such as dust, silica, and asbestos [13–15].

Extensive research has linked these injury and poor health outcomes to individual factors, as well as the conditions of work, including job demands, physical work environment and psychosocial work factors (e.g., supervisor support and worker collegiality) [7]. Construction workers' injuries and poor health have been associated with the high physical demands, prolonged exposure to awkward postures, whole body vibration, long working hours, and psychosocial hazards in the work environment [2,16].

While these factors are prevalent in the construction industry, the complex work organization of construction work provides additional challenges for implementing traditional workplace prevention programs. The hierarchical structure between the site owners, general contractors, and subcontracting companies results in a fissured workplace [17]. The dynamic nature of these worksites results in workers moving on and off the site day-to-day. In addition, construction has high workforce turnover within the company, further complicating the number of workers transitioning in and out of the workforce, which has been linked to higher injury rates [18,19].

Integrated approaches that address the work environment to improve both occupational safety and health outcomes, and worker wellbeing outcomes, are acknowledged as being the most successful [20,21]. However, these integrated and comprehensive interventions for construction worksites need further investigation [20–25]. To date, most integrated approaches for construction workers have been individual-based, or those provided through labor unions [24,26]. Many worksite-based safety interventions for construction worksites have focused on using simple campaigns (such as poster and leaflet educational material) [27,28], training programs [29,30], behavioral management programs [10,31,32], or new, task-specific ergonomic tools and methods [33,34]. The intervention, "All the Right Moves" (ARM) described in this study, tested a different approach based on integrated approaches promoted by The National Institute for Occupational Safety and Health (NIOSH) Total Worker Health*®* program. Such approaches target the conditions of work that affect workers injuries and health outcomes [7,35]. The ARM intervention targeted the conditions of work through a worksite-based ergonomics program integrated into current work practices and on-site opportunities for workers to improve their health behaviors. The project's goal was to develop and determine the feasibility of an integrated health promotion and health protection worksite-based program designed specifically for the dynamic nature of a commercial construction work site.

The purpose of this study was to examine the intervention—ARM, on commercial construction sites, using a mixed methods approach. The specific aims of this project were to examine the efficacy of an integrated program including: (1) a soft tissue injury prevention program on workers' perception of worksite ergonomic practices, new pain and injury incidences, and work limitations; and (2) a health promotion/health coaching (Health Week) program for diet, leisure time physical activity, and reduced smoking behaviors.


**Author details**

Shyam Sunder Sharma Department of Mechanical Engineering, Manipal University Jaipur, India

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