Serge Pr

Mordon

Treating hidradenitis suppurativa with photodynamic therapy

Hidradenitis suppurativa is a chronic, recurring, and disabling inflammatory condition of the skin. There is no cure for hidradenitis suppurativa and treatment must be adapted to each individual patient. Several studies have been published since 2004 on the use of photodynamic therapy to treat hidradenitis suppurativa. The use of superficial or interstitial illumination with 5-Amino-Levulinic Acid (5-ALA) or methylene blue (MB) have been proposed. Injecting 5-ALA or MB followed by illumination with a fiber optic sensor placed inside the lesion appears to be a better method of treating these thick lesions.

Introduction

Hidradenitis suppurativa is a chronic, recurring, and disabling inflammatory condition of the skin. Although we do not know exactly what causes hidradenitis suppurativa, studies suggest that it may be attributable to specific abnormalities within the hair follicles. Hidradenitis suppurativa is related to dysfunctional follicular epithelial cells and commonly affects the skin in the armpits, below the breasts, and around the groin. Hidradenitis suppurativa affects between 1% and 4% of the population in Europe and is more common in women than in men, at a ratio of 4:1.

It occurs in a number of forms, which may vary from one person to the next. Mild cases present as small lumps, blackheads, or a few cysts, while the most severe cases can take the form of multiple and recurring abscesses that may leak foul-smelling pus (Figure 1). The lesions caused by hidradenitis suppurativa can be very uncomfortable and painful, and sufferers often find their quality of life is affected.

Treating hidradenitis suppurativa

There is no cure for hidradenitis suppurativa and treatment must be adapted to each individual patient, depending on the disease severity according to Hurley staging (1) (Table 1 and figure 2). Treatment is primarily aimed at preventing new lesions, providing early and effective treatment for newly formed lesions, and removing the existing nodules and sinus tracts. Standard practice is to offer nonmedical procedures, topical and systemic treatments, and surgery.

Several studies have been published since 2004 on the use of photodynamic therapy to treat hidradenitis suppurativa. The initial studies used the same protocol as for treating actinic keratosis, i.e., topical photodynamic therapy. More recently, the use of interstitial illumination and methylene blue (MB) have been proposed.

Photodynamic therapy with topical 5-ALA

According to published articles, the technique used when treating the condition with PDT is similar to that used for actinic keratosis, i.e., MAL (Methyl AminoLevulinate)-PDT (PhotoDynamic Therapy) (MAL 16mg/g) under occlusion for 2.5 hours, and illumination with Aktilite® (PhotoCure ASA, Oslo, Norway) 37 J/cm² or Levulan© in the USA with blue light.

In this case, the incubation time is between 15 and 30 minutes and the blue light exposure lasts 18 minutes in order to reach a fluence of 10 J/cm². A summary of the various studies using topical 5-animo-levulinic acid (5-ALA) was published by Agut-Busquet et al. in 2016 [START_REF] Agut-Busquet | Photodynamic therapy with intralesional methylene blue and a 635 nm light-emitting diode lamp in hidradenitis suppurativa: a retrospective follow-up study in 7 patients and a review of the literature[END_REF]. Out of 12 articles, 8 were in favor of PDT and the other 4 did not report any response. Of these four articles, only one used blue light but with minimal penetration depth, which could explain why the treatment was not effective (3).

Unfortunately, none of the studies were randomized and patient follow-up was usually limited to only 6 months.

Intralesional photodynamic therapy (iPDT)

5-ALA penetrates skin lesions poorly when applied topically. In fact, Maisch et al. observed the formation of PpIX in the skin at a maximum depth of only 100 µm after a topical application of 5-ALA (4). It is therefore easy to see why this treatment has very limited clinical benefits for Stage II and III lesions. Valladares et al. thus suggested an intralesional injection of 1% 5-ALA in physiological serum. When injected, the 5-ALA penetrates directly into the lesions and so this is where the PpIX is produced [START_REF] Valladares-Narganes | Treatment of hidradenitis suppurativa with intralesional photodynamic therapy using a laser diode attached to an optical cable: a promising new approach[END_REF]. More recently, the same team used a 5% 5-ALA gel (Intrala 1, IDP Light SL, Alicante, Spain). Lesions were incubated under an opaque dressing for two hours prior to illumination with a 400 µm core optical fiber inserted into the lesion using a needle. This optical fiber was connected to a laser emitting 630 nm (Intermedic) at 1.2 W, therefore delivering 180 J/cm² (Figure 3). Depending on the progression of the lesion, this treatment can be repeated after 5-7 weeks [START_REF] Valladares | Treatment of hidradenitis suppurativa with intralesional photodynamic therapy with 5-aminolevulinic acid and 630 nm laser beam[END_REF].

In a series of 38 patients treated with this Intralesional photodynamic therapy (iPDT) technique between 2011 and 2015, 29 achieved a complete response. The lesion persisted in eight patients, and there was one relapse. A complete response was obtained for 68.2% of armpit lesions, 88.5% of groin lesions, 88.9% of buttock lesions and 100% for other locations (Figure 4 and5). Out of the 38 patients, 18 obtained a complete response after just one session and tolerated the treatment well [START_REF] Valladares | Treatment of hidradenitis suppurativa with intralesional photodynamic therapy with 5-aminolevulinic acid and 630 nm laser beam[END_REF].

Methylene blue

Both Fadel et al. and Agut-Busquet et al. recently conducted studies with MB (2,7). MB, which is non-toxic and inexpensive, is widely available at all hospital pharmacies because it is the first-line treatment for methemoglobinemia. It has a short incubation period of 5-20 minutes. When used in gel form, it appears to penetrate deeper into the lesions. These two teams illuminated the lesions at 635 nm. This was due to the fact that this wavelength had been developed for use with 5-ALA and was therefore available. However, the peak absorbance of MB is observed at 668 nm (8) (Figure 6).

Fadel's use of a pulsed flashlamp (EPI-C Plus; Espansione Group, Bologna, Italy) fitted with a 630 nm filter [START_REF] Mordon | Site-specific methylene blue delivery to pilosebaceous structures using highly porous nylon microspheres: an experimental evaluation[END_REF], 25 cm², 20 ms, 25 J/cm² also calls into question the action of photodynamic therapy compared to selective thermal action.

The 10 patients treated by Fadel (seven women, three men) received an average of eight sessions of IPL. The lesions beneath the breasts responded faster than those in the groin or on the buttocks. Moderate improvement was achieved for Stage III lesions (Figure 7)

In the study by Agut-Busquet et al., the photosensitizer was a 1% MB solution injected into the lesion under ultrasound guidance until the lesion turned dark blue. After a 15-minute incubation period, the lesions were illuminated with an Aktilite® device (Galderma) or BF-RhodoLED® lamp (Biofrontera) with the same parameters as for 5-ALA: 37 J/cm² for each lesion (average irradiation time of eight minutes) (Figure 8). A total of seven patients were treated: two received only one session, the other five received two sessions spaced 15 days apart. Patient follow-up was at 1, 2, 4 and 6 months.

A good response was seen in six patients after 1 month. At 6 months, 5 patients (71%) were in remission in the treated area. These preliminary results with MB are interesting, especially since the illumination parameters were suboptimal.

Figure1.

  Figure1. Main sites of hidradenitis suppurativa. (A) and (B) Armpits: multiple interconnected sinus tracts and abscesses across the entire area, on both sides, Hurley Stage III. (C) Genital area: one single abscess with a sinus tract in the fold between the pubis and penis, Hurley Stage II. (D) and (E) Groin: diffuse interconnected sinus tracts across the entire area, with severe scarring on both sides, Hurley Stage III. (Data from Lee et al.) (9)

Table 1 :Figure 2 :

 12 Figure 2: Clinical example of HS pre-PDT treatment (a) and post-PDT treatment (b) 5-ALA-PDT treatment (10).

Figure 3 :

 3 Figure 3: Diagram illustrating intralesional illumination using iPDT. (A) Irradiation following the natural path of the fistula with an intralesional fiber optic sensor. (B) Irradiation field encompassing the cavity filled with gel (brown) and inflamed tissue (pink) around the sensor (6).

Figure 4 :Figure 5 :

 45 Figure 4: Results by location of lesions (6)

Figure 6 :

 6 Figure 6: Absorption spectrum of methylene blue. The peak is at 668 nm (8)

Figure 7 :

 7 Figure 7: Stage III hidradenitis suppurativa in a male patient (A) before treatment; (B) only moderate improvement was achieved after 12 treatments due to the fibrosis and scarring (Fadel et al.) (7).

Figure 8 :

 8 Figure 8: Images of the procedure. Intralesional administration of methylene blue followed by illumination with a 635 nm light-emitting diode (Agut-Busquet et al.) (2).

Conclusion

Recent results from using iPDT to treat hidradenitis suppurativa have been highly promising.

Injecting 5-ALA or MB followed by illumination with a fiber optic sensor placed inside the lesion appears to be a better method of treating these thick lesions.


A Canovas

Gonzalo Rincon

Claudia Bevilacqua

Alma Islas-Trejo

Pauline Brenaut

Russell C Hovey

Marion Boutinaud

Caroline Morgenthaler

Monica K Vanklompenberg

Patrice Martin
email: patrice.martin@jouy.inra.fr

Angela Ca ´novas

Gonzalo Rinco

Juan F Medrano
email: jfmedrano@ucdavis.edu

Comparison of five different RNA sources to examine the lactating bovine mammary gland transcriptome

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

microdissected mammary epithelial cells (LCMEC), milk fat globules (MFG) and antibody-captured mammary epithelial cells from milk (mMEC) using RNA-Sequencing.

Results

RNA preparation and amplification. A total of 14 bovine samples (three MGT, three LCMEC, three MFG, two SC and three mMEC from two Jersey and one Normande cows were analyzed by RNA-Sequencing. Figure 1 shows the chromatogram traces profiling the quality of the RNA obtained for each fraction. The spectrophotometric absorbance profile for RNA from the MGT (Figure 1A) and LCMEC (Figure 1C) were similar (RIN 5 6.5-7.0). The quality of the RNA may be affected by time taken to obtain the tissue biopsy until it is frozen, and the time to cut the sections to freeze in the OCT blocks used for LCMEC. Comparison between mammary gland epithelial cells before and after laser microdissection was performed (Figure 2). Alveolar (acinar) structures (Figure 2A) lined by mammary epithelial cells (yellow arrows) could be easily distinguished after staining mammary tissue sections with Mayer's hematoxylin (Sigma). The LCMEC were dissected and collected on a cap at 603 (Figure 2B1) and 43 (Figure 2B2) magnifications. The RIN was similar (6.5 to 7.0) between MGT (Figure 2A1) and LCMEC (Figure 2B3), as illustrated in the chromatogram profiles. The total RNA yield for one laser capture cap ranged between 5 to 10 ng. In addtion, to assess a possible contamination of microdissected MEC, qPCR systems were carried out to quantify specific markers for mammary myoepithelial cells (KRT14), lymphocytes (CD3e), neutrophils (CD18), macrophages (CD68 and CD18) and MEC (CSN3). In general, LCMEC showed a similar profile for each sample, with overexpression of MEC specific markers (LALBA and CSN2S2) and under-expression of mammary myoepithelial cells markers (KRT14) and immune cells markers (CD3e and CD68). Also, the same profile was observed in MFG and mMEC fractions with an over-expression of marker CD68, suggesting the presence of macrophages (Supplementary File 2).

High RIN values (around 8.0) were consistently observed for total RNA from SC (Figure 1B) despite the fact that the milk sample was kept on ice for three hour until transported to the laboratory for RNA extraction. The RNA from MFG contains low molecular weight fragments and a very low amount of ribosomal RNA (RIN 5 6.0) (Figure 1E).

The membrane of MFG contains specific components such as phospholipids, proteins and glycophospholipids that are known to have an affinity for the surface of bacterial cells. By analyzing all the MFG reads not mapping to the bovine genome (,11 million reads), we identified the presence of 618 different bacteria. In contrast, approximately 9 million not mapped reads were found in SC as being associated just with E. coli.

The mMEC samples purified with the antibody method presented a pattern similar to the MFG RNA, with a large fraction of low molecular weight RNA (Figure 1D) significantly impacting the RIN value (RIN 5 6.0). The quality of RNA obtained from the mMEC in this study was not representative of the quality generally obtained with this method [START_REF] Boutinaud | Milking and feed restriction regulate transcripts of mammary epithelial cells purified from milk[END_REF] .

RNA-Seq expression analyses. An average of 32 million shortsequence reads was obtained for each individual sample; these were assembled and mapped to the annotated UMD3.1 bovine genome assembly (27,368 genes). In all the samples, 60-75% of the reads were categorized as mapped to the bovine reference sequence (Table 1).

RPKM values [START_REF] Mortazavi | Mapping and quantifying mammalian transcriptomes by RNA-Seq[END_REF] were used to establish the total number of genes expressed in the MGT, SC, LCMEC, MFG and mMEC transcriptome. In this context, approximately 90% of the total annotated genes in the UMD3.1 bovine genome assembly were expressed in the samples under study (24,616 genes out of a total of 27,368).

Correlations between RPKM expression levels for the MGT, SC, LCMEC, MFG and mMEC transcriptomes. The different sampling methods were compared by calculating a Pearson correlation between the RPKM expression levels of the ,24,616 expressed genes in samples of MGT, SC, LCMEC, MFG and mMEC. Our results indicate that gene expression is highly correlated among Chromatograms from total RNA capillary electrophoresis; MGT before microdissection (A1) LCMEC after microdissection (B3).

LCMEC, MGT, SC and MFG. These sampling methods showed the highest correlation values in gene expression with LCMEC, ranging from r 5 0.80 to r 5 0.99 (MGT), from r 5 0.80 to r 5 0.95 (SC) and from r 5 0.81 to r 5 0.93 (MFG) in Jersey and Normande cows, respectively (Table 2). Interestingly, gene expression in SC was significantly correlated with that in MGT in Jersey and Normande cows (r 5 0.98 and r 5 0.92, respectively). By contrast, a low correlation existed between mMEC and LCMEC in both Jersey and Normande cows (correlation ranged from r 5 0.15 to r 5 0.39, Table 2). In addition, independent correlation analysis were performed using the list of highly expressed ($500 RPKM), normally/medium expressed (ranged from 500 to 10 RPKM) and lowly expressed (,10 RPKM) genes [START_REF] Wickramasinghe | Transcriptional profiling of bovine milk using RNA sequencing[END_REF] . A homogeneous distribution of expressed genes was obtained across LCMEC, MGT, SC, MFG and mMEC. All fractions had a higher number of lowly expressed genes. The high correlation among LCMEC, MGT, SC and MFG persist in highly-expressed, normally/medium-expressed and lowly-expressed genes (Additional files 3, 4 and 5).

A correlation was also calculated using the ranking/position of the RPKM gene expression values. The ranking/position stretched from the most highly expressed to the lowest expressed gene for each sample analyzed. Consistently, the highest correlated values were between LCMEC and MGT, MFG and SC samples in both breeds (correlation ranged from r 5 0.90 to r 5 0.94; p , 0.0001). In this analysis, relatively high correlations (ranging from r 5 0.79 to r 5 0.90) were observed between LCMEC and mMEC (Supplementary File 6).

Most highly expressed genes in MGT, SC, LCMEC, MFG and MEC. We also compared the genes having the greatest expression in MGT, SC, LCMEC, MFG and mMEC (Table 3). As reported by Wickramasinghe et al. [START_REF] Wickramasinghe | Transcriptional profiling of bovine milk using RNA sequencing[END_REF] for milk SC, the genes having the greatest expression in all MGT, SC, LCMEC and MFG transcriptomes were the caseins (i.e. CSN2, CSN3, CSN1S1 and CSN1S2), the main two whey proteins (BLG and LALBA) and GLYCAM1 (glycosylationdependent cell adhesion molecule 1). Among these, CSN2 and CSN3 showed the highest RPKM values ranging from 207,305 to 1,877 depending on the sample (Table 3). BLG, LALBA and GLYCAM1 were abundantly expressed in MGT, SC, LCMEC and MFG. Surprisingly BLG, LALBA and GLYCAM1 were not highly expressed in mMEC. In general the overall transcriptome expression level in mMEC was several orders of magnitude lower than in the other samples. In cattle, mMEC only account for no more than 5% of the somatic cells [START_REF] Wagner | Effect of endotoxic mastitis on epithelial cell numbers in the milk of dairy cows[END_REF] . Also, the additional step in the purification of mMEC from SC may explain the observed different expression profile.

A comparison of the list of the most highly expressed genes revealed that approximately 15 out of the 17 genes with greatest levels of expression in LCMEC were also expressed abundantly in both MGT and SC. In contrast, only 9 out of these 17 were abundantly expressed in mMEC (Table 3). The transcriptome for MGT, SC, LCMEC and MFG had a very similar expression pattern. Both SC and MFG showed highly expressed genes, such as SPP1 (secreted phosphoprotein 1 or osteopontin), RPS17 (ribosomal protein S17), TCTP (translationally controlled tumor protein) and PTMA (prothymosin alpha). Among these, SPP1 cooperates with other pro-inflammatory cytokines such as TNFa to participate in the overall inhibition of mammary epithelial cell viability that is required for tissue remodeling in the bovine mammary gland during the course of involution [START_REF] Piantoni | Expression of metabolic, tissue remodeling, oxidative stress, and inflammatory pathways in mammary tissue during involution in lactating dairy cows[END_REF] . Furthermore, MFG showed high levels of gene expression for perilipin 2 (PLIN2) that is related to lipid accumulation and secretion [START_REF] Ambrosio | The alteration of lipid metabolism in Burkitt lymphoma identifies a novel marker: adipophilin[END_REF] . In contrast, mMEC showed a different profile for all caseins and major whey proteins, and also uniquely expressed high levels of the TUBA1B (tubulin alpha 1B), HMOX1 (heme oxygenase (decycling) 1), S100A11 (S100 calcium binding protein A11) and RL10 (ribosomal protein L10) genes in Jersey cows. These genes represent markers of cell development and immunity. Also, expression of CD68 (CD68 molecule), a specific marker for macrophages, was elevated in the SC and mMEC transcriptomes in Jersey cows, perhaps due to the presence of a higher proportion of macrophages.

Discussion

The transcriptome for mammary gland was studied using five different sampling methods. Total RNA from MGT, SC, LCMEC, MFG and mMEC were compared using RNA-Sequencing to examine the representation of genes and their levels of expression.

The integrity and type of the RNA varied considerably between samples depending on the sampling method, which in turn had a bearing on the RNA-Sequencing results (Table 1). The RNA from the MGT, LCMEC and SC showed similar electrophoresis chromatograms profiles. The RNA from MFG contained low molecular weight fragments and a very low amount of ribosomal RNA due to the presence of small amounts of cytoplasmic material in the bovine milk fat globules during the MFG secretory process [START_REF] Huston | Factors related to the formation of cytoplasmic crescents on milk fat globules[END_REF] . This likely explains the lower RIN value (RIN 5 6.0) of this tissue. The low MW RNA may be due to the presence of bacteria and also very small RNAs that are abundant in this fat fraction [START_REF] Munch | Transcriptome profiling of microRNA by Next-Gen deep sequencing reveals known and novel miRNA species in the lipid fraction of human breast milk[END_REF] . The MFG membrane contains specific components such as phospholipids, proteins and glycophospholipids that are unique in this cell fraction. Interestingly, these components are known to have an affinity for the cell surface of bacteria [START_REF] Brisson | Characterization of Lactobacillus reuteri interaction with milk fat globule membrane components in dairy products[END_REF] . Considering this, we analyzed all the MFG reads not mapping to the bovine genome (,11 million reads) for the presence of bacterial genes. From this analysis we found that 618 different kinds of bacteria were identified with MFG. Comparatively, approximately 9 million reads were found in SC as being associated just with E. coli. The mMEC samples purified with the antibody method presented a pattern similar to the MFG RNA, with a large fraction of low molecular weight RNA significantly impacting the RIN value (RIN 5 6.0).

The transcriptome for bovine LCMEC was highly correlated with that for MGT, SC and MFG, and showed a high correlation with MGT in Jersey (r 5 0.80) and Normande (r 5 0.99) cows (p , 0.0001). A significant correlation was found between MGT and SC in Jersey (r 5 0.98) and Normande (r 5 0.92) cows. By contrast, a low correlation existed between mMEC and LCMEC in both Jersey and Normande cows (correlation ranged from r 5 0.15 to r 5 0.39). The MGT is complex and heterogeneous, with multiple cell types including myoepithelial, stromal and immune cells. In terms of immune cells, SC is more similar to MGT than mMEC due to the high proportion of immune cells such as neutrophils, lymphocytes and macrophages which account for ca. 95% of SC in milk from healthy cows [START_REF] Wagner | Effect of endotoxic mastitis on epithelial cell numbers in the milk of dairy cows[END_REF] . It is important to note that the isolation of mMEC utilized the binding of bovine MEC to a monoclonal antibody recognizing cytokeratins [START_REF] Boutinaud | Milking and feed restriction regulate transcripts of mammary epithelial cells purified from milk[END_REF] . Different antibody reactions both with not much high RNA quality level can alter and affect the final product, which may explain why these samples had a low correlation with all the cellular fractions analyzed (range from r 5 0.13 to r 5 0.50).

In general, the highly expressed genes such as caseins (CSN1S1, CSN1S2, CSN2, CSN3) and whey proteins (BLG, LALBA) showed a similar expression profile in MGT, LCMEC, SC and MFG. However, MFG had a unique abundant expression of the PLIN2 gene that is implicated in lipid accumulation. The majority of the lipid produced during lactation is secreted into milk by a process of membrane envelopment of cytoplasmic lipid droplets (CLDs). Others have hypothesized that PLIN2 plays a pivotal role in both formation and secretion of milk lipids, where it functions as an adaptor to couple CLDs to the cytoplasmic leaflet of the apical plasma membrane of MEC [START_REF] Chong | Determinants of adipophilin function in milk lipid formation and secretion[END_REF] . This gene may also be involved in development and maintenance of adipose tissue. Furthermore, PLIN2 is expressed by a wide range of cultured cell lines, including fibroblasts, endothelial and epithelial cells, and tissues, suggesting that it may serve as a marker of lipid accumulation in diverse cell types and diseases [START_REF] Heid | Adipophilin is a specific marker of lipid accumulation in diverse cell types and diseases[END_REF] .

Expression of CD68 (CD68 molecule), a specific marker for macrophages, was elevated in the SC and mMEC transcriptomes in Jersey cows, perhaps due to the presence of a higher proportion of macrophages. This finding would suggest that mMEC purification was not totally effective. Although the antibody-directed mMEC purification method allowed the specific selection of epithelial cells, which account for less than 5% of total milk cells (SC), the mMEC transcriptome was different from the rest of the transcriptomes analyzed. In our results, immunomagnetic mMEC purification method showed that a conjoint of reactions was affecting the proportion and/ or the function of white cells present in milk, showing a unique set of highly expressed genes such as TUBA1B, HMOX1, S100A11 and RL10 involved in anti-inflammatory processes, cell death or apoptosis, specifically in Jersey mMEC samples.

Up to this point, the majority of gene expression analyses related to bovine lactation have been developed using tissues obtained by biopsy [START_REF] Finucane | Onset of lactation in the bovine mammary gland: gene expression profiling indicates a strong inhibition of gene expression in cell proliferation[END_REF][START_REF] Vanklompenberg | Technical note: A vacuum-assisted approach for biopsying the mammary glands of various species[END_REF] . This biopsy approach is invasive during a stage when vascularization of the gland is at a maximum, which presents significant technical challenges and management issues in the recovery of the animals.

An alternative sampling procedure has been proposed via isolating mRNA directly from mMEC [START_REF] Boutinaud | Milking and feed restriction regulate transcripts of mammary epithelial cells purified from milk[END_REF][START_REF] Ben Chedly | Use of milk epithelial cells to study regulation of cell activity and apoptosis during once-daily milking in goats[END_REF] , MFG [START_REF] Brenaut | Validation of RNA isolated from milk fat globules to profile mammary epithelial cell expression during lactation and transcriptional response to a bacterial infection[END_REF] and SC [START_REF] Boutinaud | Use of somatic cells from goat milk for dynamic studies of gene expression in the mammary gland[END_REF][START_REF] Feng | Extraction and quantitative analysis of stearoyl-coenzyme A desaturase mRNA from dairy cow milk somatic cells[END_REF] that are released into milk during lactation. Using RNA extracted from mMEC, MFG or SC represents a more accessible method for assessing gene expression in the mammary epithelium [START_REF] Boutinaud | Potential uses of milk epithelial cells: a review[END_REF] , particularly when dynamic studies require the repeated analysis of samples from the same animal. Considering the cost and labor involved to obtain a sample of mammary parenchyma for RNA extraction, analyzing milk SC and MFG appears to be an alternative, direct and simple approach that provides a high correlation with gene expression in MGT and LCMEC without the need to perform a biopsy of the mammary gland tissue. Our results present the similarities between different sampling methods by examining the transcriptome of mammary gland tissue and different RNA sources from milk. This information can guide investigators in choosing the most appropriate sampling method to examine specific physiological states from different sources of RNA during lactation for different research applications. One of the simplest procedures to examine the transcriptome associated with milk appears to be to isolate mRNA directly from MFG and SC that are released into milk during lactation. Our analyses suggest that the SC and MFG transcriptome are the most representative of MGT and LCMEC and can be used as an effective and easy way to study gene expression using RNA-Sequencing, without the need to perform a mammary gland biopsy.

Figure 1 |

 1 Figure 1 | Total RNA capillary electrophoresis chromatograms from mammary and milk fractions. (A) Mammary tissue (MGT), (B) Somatic cells (SC), (C) Laser capture mammary epithelial cells (LCMEC), (D) Antibody-captured milk mammary epithelial cells (mMEC), (E) Milk fat globule (MFG). Traces (A) and (C) (Agilent Bioanalyzer), traces (B), (D), (E) Experion Analyzer (BioRad). These diagrams show the differences in quality of RNA obtained from the different fractions.

Figure 2 |

 2 Figure 2 | Mammary gland tissue (MGT) before microdissection (A) and mammary epithelial cells after laser microdissection (LCMEC) (B1 and B2).

Table 1 |

 1 Total reads and mapped reads obtained in each sample analyzed Reads categorized as mapped to the annotated UMD3.1 bovine genome assembly (27,368 genes). MGT: mammary gland tissue; SC: milk somatic cells; LCMEC: laser microdissected mammary epithelial cells; MFG: milk fat globules; mMEC: antibody-captured milk mammary epithelial cells.

	RNA Fraction	Sample number	Breed	Reads (n)	*Mapped reads (n)	% mapped reads
	MGT	653	Jersey	40,147,740	24,732,197	61.6
		671	Jersey	38,508,068	23,163,085	60.2
		1816	Normande	26,480,391	19,732,280	74.5
	SC	653	Jersey	40,209,699	28,036,287	69.7
		671	Jersey	39,507,994	26,136,100	66.2
	LCMEC	653	Jersey	27,335,082	17,121,159	62.6
		671	Jersey	24,355,141	14,345,582	58.9
		1816	Normande	23,173,807	16,973,772	73.3
	MFG	653	Jersey	23,918,861	14,588,022	60.9
		671	Jersey	24,334,219	14,475,382	59.5
		1816	Normande	34,023,717	22,407,126	65.9
	mMEC	653	Jersey	38,899,626	29,284,686	75.3
		671	Jersey	38,495,029	28,292,867	73.5
		1816	Normande	29,191,918	22,056,958	75.6
	Average			32,041,521	21,524,679	66.9

*

Table 2 |

 2 Correlations between RPKM expression levels of MGT, SC, LCMEC, MFG and mMEC transcriptome in Jersey and Normande cows

				MGT	SC		LCMEC		MFG		mMEC
			Jersey	Normande	Jersey	Jersey	Normande	Jersey	Normande	Jersey	Normande
	MGT	Jersey	1.00	-	-	-	-	-	-	-	-
		Normande	0.97	1.00	-	-	-	-	-	-	-
	SC	Jersey	0.98	0.92	1.00	-	-	-	-	-	-
	LCMEC	Jersey	0.80	0.90	0.79	1.00	-	-	-	-	-
		Normande	0.90	0.99	0.95	0.80	1.00	-	-	-	-
	MFG	Jersey	0.90	0.88	0.93	0.81	0.85	1.00	--	-	-
		Normande	0.88	0.92	0.95	0.85	0.93	0.93	1.00	-	-
	mMEC	Jersey	0.18	0.15	0.26	0.39	0.23	0.50	0.37	1.00	-
		Normande	0.15	0.16	0.22	0.23	0.15	0.37	0.32	0.67	1.00

MGT: mammary gland tissue; SC: milk somatic cells; LCMEC: laser microdissected mammary epithelial cells; MFG: milk fat globules; mMEC: antibody-captured milk mammary epithelial cells.

Table 3 |

 3 Most highly expressed genes in MGT, SC, LCMEC, MFG and mMEC transcriptome in Jersey and Normande cows expressed in RPKM values (reads per kilo base per million mapped reads). Green color represents the most highly expressed genes, red color represents the lowest expressed genes and the different color tones are intermediate values : mammary gland tissue; SC: milk somatic cells; LCMEC: laser microdissected mammary epithelial cells; MFG: milk fat globules; mMEC: antibody-captured milk mammary epithelial cells. Jersey data provided correspond to mean values of the two analyzed Jersey cows.

			MGT	SC		LCMEC		MFG		mMEC
	Gene	Jersey	Normande	Jersey	Jersey	Normande	Jersey	Normande	Jersey	Normande
	CSN2	207305	145263	156130	105065	179820	76891	45571	13651	2591
	CSN3	72233	87335	30208	51838	84031	15602	18689	1877	1991
	CSN1S1	39736	45340	24903	25982	50297	11528	7652	956	710
	CSN1S2	12056	32973	4010	9093	29740	2156	4592	206	551
	LALBA	5619	6537	3056	2706	11139	2693	1719	43	42
	GLYCAM1	4035	8346	3579	2668	10633	1758	2995	119	90
	BLG	3231	2407	6835	2697	5925	3854	707	121	84
	SPP1	1406	1036	6975	845	1301	927	6250	10680	992
	RPS17	1374	366	3164	120	90	2183	916	118	69
	TCTP	937	1098	2724	974	917	2451	2656	1320	42
	PTMA	893	123	2876	401	104	1843	392	3345	28
	MFGE8	705	186	576	75	84	95	49	177	2
	CD36	486	691	264	322	673	56	112	65	27
	SCD	265	459	44	222	570	39	98	4	6
	FABP4	171	527	138	182	103	90	123	5	4
	FRIH	127	134	1898	121	87	779	3400	2165	53
	RL19	519	358	1351	348	405	1142	717	255	16
	RS12	412	152	1172	516	186	66	66	1379	84
	CD68	3	3	1108	7	4	84	62	1755	46
	RS17	157	117	117	1577	433	87	119	1499	33
	B2MG	171	156	766	482	265	97	1055	84	21
	PLIN2	57	65	77	75	73	994	8195	38	23
	TUBA1B	46	39	38	35	37	59	46	1625	53
	HMOX1	4	0.2	1	0.09	0.04	7	3	1073	54
	S100A11	33	4	2	10	5	51	10	936	57
	RL10	36	4	34	32	35	45	33	668	53

MGT

SCIENTIFIC REPORTS | 4 : 5297 | DOI: 10.1038/srep05297

Acknowledgments

The authors would like to thank Doug Gisi from U.C. Davis Dairy Facility and S. Leurent and Y. Gallard from INRA Experimental Farm of Le Pin-au-Haras, France and S. Marion from UMR PEGASE for their technical help. We gratefully acknowledge the support for this collaborative project from the France-Berkeley Fund.

Methods

Animal material. Mammary gland tissue and milk samples were obtained from two Jerseys and one Normande cow at the University of California -Davis (UCD) and the Institut National de la Recherche Agronomique (INRA) herd (Le Pin-au-Haras, France), respectively. Jersey cows were in their 3 rd and 4 th lactation around 100 days in milk whereas Normande cow was in its 2 nd lactation around 180 days in milk. Mammary tissue biopsies were performed using a 9-gauge, 12-cm probe with a 12mm aperture as described by VanKlompenberg et al. [START_REF] Vanklompenberg | Technical note: A vacuum-assisted approach for biopsying the mammary glands of various species[END_REF] in Jersey cows and 70 3 4 mm rotating stainless steel cannula in Normande cow [START_REF] Farr | An improved method for the routine biopsy of bovine mammary tissue[END_REF] . A 5-mm incision was made through the skin and connective tissue approximately half-way up the rear udder quarter using a scalpel blade before the parenchyma was penetrated with a Hologic ATEC Biopsy Console Handpiece for excision of a tissue core (Hologic Inc., Bedford, MA) and Farr's Instrument. Triplicate milk samples were collected before performing tissue biopsies and were held on ice. Sample collections and procedures were performed in accordance with the approved guidelines of the UC Davis Institutional Animal Care and Use Committee (IACUC).

RNA extraction. Biopsied mammary gland tissue (MGT) was divided in two: one part was immediately put in a cryomold with OCT compound and frozen with a mix of dry ice and isopropanol for future microdissection. The other portion was frozen in liquid nitrogen for subsequent extraction of RNA. Total RNA from MGT was extracted using Trizol (Invitrogen).

Laser capture microdissection of epithelial cells (LCMEC) from the mammary tissue was performed using an Arcturus Veritas Microdissection system and software (Applied Biosystems). Cryosectioning, staining, dehydration and harvesting of microdissected cells was performed as described Bevilacqua et al. [START_REF] Bevilacqua | Maintaining RNA integrity in a homogeneous population of mammary epithelial cells isolated by Laser Capture Microdissection[END_REF] . Total RNA was extracted from captured cells using the PicoPureH RNA Isolation Kit (Arcturus, Applied Biosystems) according to the manufacturer's instructions, including an oncolumn DNase I treatment (Qiagen). An aliquot of RNA was reverse transcribed to validate the selectivity and specificity of laser capture microdissection by qPCR for markers of myoepithelial cells, MEC and immune cells. To assess a possible contamination of microdissected MEC, qPCR systems designed to quantify specific markers for mammary myoepithelial cells (Krt14), lymphocytes (CD3e), neutrophils (CD18), macrophages (CD68 and CD18) and MEC (CSN3) were implemented. Sequences were from GenBank (Supplementary File 1).

Milk was centrifuged at 2,000 g for 10 min to pellet somatic cells (SC) and to separate the upper milk fat globule (MFG) fraction. The MFG fraction was mixed with Trizol LS and heated briefly at 30uC while shaking to emulsify the fat. Total RNA was extracted from milk somatic cells (SC) and from milk fat using Trizol (Invitrogen) following the protocol from the manufacturer as described in Ca ´novas et al. [START_REF] Ca ´novas | SNP discovery in the bovine milk transcriptome using RNA-Seq technology[END_REF] and Brenaut et al. [START_REF] Brenaut | Validation of RNA isolated from milk fat globules to profile mammary epithelial cell expression during lactation and transcriptional response to a bacterial infection[END_REF] , respectively. In addition, MEC from milk (mMEC) were obtained according to the procedure described by Boutinaud et al. [START_REF] Boutinaud | Milking and feed restriction regulate transcripts of mammary epithelial cells purified from milk[END_REF] using a protocol based on the binding of mMEC to a monoclonal antibody recognizing cytokeratin 8 (clone 34BE12, Sigma-Aldrich), except that milk was kept on ice for approximately 3 h until it was transported to the laboratory. In all cases the purified total RNA was treated with Turbo DNase (Invitrogen, Carlsbad, CA). Quality of the total RNA was evaluated using the RNA Integrity Number (RIN) value in the Agilent Bioanalyzer 2100 or in the Experion Analyzer (BioRad).

RNA-Sequencing. RNA-Sequencing was used to measure gene expression in the MGT, SC, LCMEC, MFG and mMEC transcriptome. Total RNA (DNAse-treated) was used to synthesize amplified cDNA following the protocol in the Ovation RNAseq system and the Encore NGS library system (NuGEN, San Carlos, CA). Total RNA (100 ng DNase treated) was subjected to an RNA amplification process using the Ribo-SPIA technology from the Ovation RNA-seq System (NuGEN, San Carlos, CA). The amplified cDNA (200 ng) was fragmented using the Bioruptor sonicator (Diagenode) 30 sec ON, 30 sec OFF cycles for 10 min to produce fragments with 150-200 bp size distribution. Adaptor molecules were ligated to the ends of each fragment and amplified by PCR to produce the final library using the Encore NGS library system (NuGEN, San Carlos, CA). A single library was made for each sample.

Fragmented DNA was sequenced on a GAII sequencer analyzer (Illumina, San Diego, CA).

Quality control (QC) analysis was performed using the application NGS quality control tool of CLC Genomics workbench software (CLC Bio, Aarhus, Denmark). This tool assess sequence quality indicators based on the FastQC-project (http:// www.bioinformatics.babraham.ac.uk/projects/fastqc/). Quality was measured taking into account sequence-read lengths and base-coverage, nucleotide contributions and base ambiguities, quality scores as emitted by the base caller and over-represented sequences. All the samples analyzed passed all the QC parameters having the same length (36 bp), 100% coverage in all bases, 25% of A, T, G and C nucleotide contributions, 50% GC on base content and less than 0.1% over-represented sequences, indicating a very good quality.

Short sequence single reads (36 bp) were mapped to the annotated UMD3.1 bovine reference genome (http://www.ncbi.nlm.nih.gov/genome/?term5bos1taurus). Analysis of RNA-Seq expression data was performed using the transcriptomics analysis tool of CLC Genomics workbench software. Data were normalized by calculating the 'reads per kilobase per million mapped reads' (RPKM) for each gene [START_REF] Mortazavi | Mapping and quantifying mammalian transcriptomes by RNA-Seq[END_REF] . To select expressed genes a threshold of RPKM $ 0.2 was used [START_REF] Wickramasinghe | Transcriptional profiling of bovine milk using RNA sequencing[END_REF] .

Large gap mapping analysis was performed to identify those reads not mapping to the bovine genome. Then, de novo assembly analysis was performed from unmapped reads to generate contigs using CLC Genomics workbench software (CLC Bio, Aarhus, Denmark). Blast2GO software (http://www.blast2go.com) was used for contig annotation and bacteria identification (Gotz et al., 2008). Blats2Go performs the Blast step by communicating directly to NCBI (National Center for Biotechnology Information) blast service. The Blastn algorithm was used to compare a nucleotide query sequence against all the nucleotide sequence database. A statistical significance threshold of 1.0 E-3 (e-value) was established for reporting matches against the database sequences. Also a minimal length (30 bp) of a blast hit and 90% of similarity was used to exclude hits with only small local alignments from the blast results.

Statistical analysis. To examine the similarities between the different sampling procedures a correlation between the RPKM expression levels of MGT, SC, LCMEC, MFG and mMEC transcriptomes was performed using the CORR procedure of SAS (SAS Institute Inc.). This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder in order to reproduce the material. To view a copy of this license, visit http:// creativecommons.org/licenses/by-nc-nd/4.0/

Author contributions


K Prabakaran

M Jayasakthi

S Surender

S Pradeep

S Sanjay

R Ramesh

M Balaji

Nicolas Gautier

K Baskar

Structural, morphological, optical and electrical characterization of InGaN/ GaN MQW structures for optoelectronic applications

InGaN/GaN multiple quantum well (MQW) structures were grown on c-plane sapphire substrate using metal organic chemical vapour deposition technique by varying the MQW periods. The indium composition and thickness were estimated using high-resolution X-ray diffraction. InGaN well, GaN barriers and Indium composition were estimated as 3 nm, 18 nm and 16-18% using epitaxy smooth fit software. Reciprocal space mapping revealed that InGaN/GaN MQW samples were coherently strained. High-resolution transmission electron microscopy images confirmed good interface between the InGaN/GaN MQW structures. Atomic force microscopy and scanning electron microscopy exhibit decrease in the surface roughness with increase in the number of InGaN/GaN MQW periods with respect to the number of defects comprising of threading dislocations and hexagonal V-pits. Self-organized In(Ga)N like nanostructures with spiral growth mechanism was also observed due to the low temperature growth of p-GaN layer. The photoluminescence spectra of the MQWs showed a red-shift when the number of QW periods was increased due to quantum confined stark effect. Hall Effect measurement displayed good semiconducting behavior in the InGaN/GaN MQW structures. The carrier concentration values also emphasized adequate variations when number of periods was increased.

Introduction

Group III-nitride semiconductors have been an excellent candidate for various applications such as optoelectronics and high power devices [START_REF] Damilano | Yellow-red emission from (Ga, In) N heterostructures[END_REF][START_REF] Matioli | High internal and external quantum efficiency InGaN/GaN solar cells[END_REF][START_REF] Farrell | Effect of intentional p-GaN surface roughening on the performance of InGaN/GaN solar cells[END_REF][START_REF] Yang | Enhancement of the conversion efficiency of GaN-based photovoltaic devices with AlGaN/InGaN absorption layers[END_REF]. They are widely preferred for their outstanding tunable direct bandgap characteristics from near infrared (~0.7 eV-InN) to ultraviolet region (GaN-3.4 eV) by varying the Indium (In) compositions [START_REF] Farrell | High quantum efficiency InGaN/GaN multiple quantum well solar cells with spectral response extending out to 520 nm[END_REF][START_REF] Seo | Improved photovoltaic effects in InGaN-based multiple quantum well solar cell with graphene on indium tin oxide nanodot nodes for transparent and current spreading electrode[END_REF][START_REF] Jeng | Increasing solar efficiency of InGaN/GaN multiple quantum well solar cells with a reflective aluminum layer or a flip-chip structure[END_REF][START_REF] Seo | Efficiency improvement in InGaN-based solar cells by indium tin oxide nanodots covered with ITO films[END_REF].

Comparing with the other semiconducting materials, InGaN attracts more attention due to its high carrier mobility, superior radiation resistance and high optical absorption (∼10 5 cm -1 ) [START_REF] Farrell | High quantum efficiency InGaN/GaN multiple quantum well solar cells with spectral response extending out to 520 nm[END_REF][START_REF] Dahal | InGaN/GaN multiple quantum well concentrator solar cells[END_REF][START_REF] Pantzas | Semibulk InGaN: a novel approach for thick, single phase, epitaxial InGaN layers grown by MOVPE[END_REF][START_REF] Cai | Improved photovoltaic performance of InGaN/GaN solar cells with optimized transparent current spreading layers[END_REF][START_REF] Sang | Enhanced performance of InGaN solar cell by using a super-thin AlN interlayer[END_REF]. InGaN based optoelectronics devices possess excellent physical and chemical stability that allows them to operate under harsh environments [START_REF] Valdueza-Felip | Photovoltaic response of InGaN/GaN multiple-quantum well solar cells[END_REF][START_REF] Sang | InGaN-based thin film solar cells: epitaxy, structural design, and photovoltaic properties[END_REF]. In general, InGaN/GaN multiple quantum well (MQW) structures act as active regions for light emitting diode (LED), solar cells and thermoelectric applications. However, extending the emission range (From green to red) and improve the luminescence property of InGaN is still challenge [START_REF] Woo | Epitaxial growth of low temperature GaN using metal migration enhanced epitaxy for high-quality InGaN/GaN heterojunctions[END_REF]. Furthermore, to minimize the lattice mismatch between InN and GaN to obtain InGaN layer with high crystalline quality is another challenge [START_REF] Seo | Improved photovoltaic effects in InGaN-based multiple quantum well solar cell with graphene on indium tin oxide nanodot nodes for transparent and current spreading electrode[END_REF][START_REF] Mahala | Fabrication and characterization of GaN/InGaN MQW solar cells[END_REF][START_REF] Xiao-Bin | InGaN/GaN multiple quantum well solar cells with an enhanced open-circuit voltage[END_REF]. In order to surmount this intricacy, the InN layers are generally grown at low temperature (LT) due to the thermal decomposition of InN. The growth of thick InGaN layer with comparatively high indium composition is one of the critical challenges in fabricating InGaN-based optoelectronic devices [START_REF] Tsai | Enhanced power conversion efficiency in InGaN-based solar cells via graded composition multiple quantum wells[END_REF]. To overcome these issues, the In-rich thin InGaN layer based MQW structure was used as an alternative. The crystalline quality depends on various parameters such as optimization of barrier, well thickness and growth temperature of MQWs [START_REF] Xiao-Bin | InGaN/GaN multiple quantum well solar cells with an enhanced open-circuit voltage[END_REF][START_REF] Bai | Efficiency enhancement of InGaN/GaN solar cells with nanostructures[END_REF] along with the In composition of InGaN wells and number of MQW periods. However, the collection of carriers in strained MQWs can be affected considerably by the spontaneous and piezoelectric polarization fields. This phenomenon is known as quantum confinement stark effect (QCSE). It leads to the spatial separation of electron and hole wave functions, which restricts the carrier recombination efficiency [START_REF] Su | Suppressed quantum-confined Stark effect in InGaN-based LEDs with nano-sized patterned sapphire substrates[END_REF]. In addition, surface topography of Inrich InGaN based MQW structures aggravates the optical and electrical properties, through high density of structural defects like threading dislocations (TD), V-defects and trench defects during the epitaxial process [START_REF] Sheen | V-pits as barriers to diffusion of carriers in InGaN/GaN quantum wells[END_REF][START_REF] Massabuau | Correlations between the morphology and emission properties of trench defects in InGaN/GaN quantum wells[END_REF][START_REF] Kumar | Surface morphological studies of green InGaN/GaN multi-quantum wells grown by using MOCVD[END_REF][START_REF] Tsai | Efficiency enhancement of blue light emitting diodes by eliminating V-defects from InGaN/GaN multiple quantum well structures through GaN capping layer control[END_REF]. This also affects the interface of InGaN/GaN MQWs during the growth of p-GaN layers. To avoid such degradation in the quality of InGaN/GaN MQWs, the p-GaN layer is grown at lower temperatures [START_REF] Farrell | Effect of intentional p-GaN surface roughening on the performance of InGaN/GaN solar cells[END_REF][START_REF] Arif | Role of V-pits in the performance improvement of InGaN solar cells[END_REF]. A better understanding of structural, morphological, optical and electrical properties of InGaN/GaN MQW structure are necessary to achieve sharp interfaces and atomically flat surface, which enhances the efficiency [START_REF] Kumar | Surface morphological studies of green InGaN/GaN multi-quantum wells grown by using MOCVD[END_REF]. In the present work, the effects of increasing the number of periods in MQWs and the effect of growing p-GaN layer at low temperature in InGaN/GaN MQW structures have been carried out.

Experimental procedure

InGaN/GaN MQW structures were grown on 2-inch c-plane sapphire substrate using metal organic chemical vapour deposition (MOCVD, Aixtron200/4RF-S) by varying the period of MQWs. Trimethylgallium (TMGa), Trimethylindium (TMIn) and ammonia (NH 3 ) were used as precursors for gallium, indium and nitrogen respectively. Triethylgallium (TEGa) was used as the gallium precursor during the growth of barrier layer in the active region of the structures. Silane and bis-cyclopentadienyl magnesium (Cp 2 Mg) were used as n and p-type dopants respectively. High purity hydrogen (H 2 ) and nitrogen (N 2 ) were used as carrier gases. Prior to the growth, the sapphire substrates were thermally cleaned under H 2 ambience at 1050 °C for 10 min to remove residues and impurities on the surface. Subsequently, 25 nm thick GaN nucleation layer (NL) was grown at 520 °C with a V/III ratio of 3453. GaN NL was recrystallized at 1000 °C for 1 min. The growth temperature was ramped up to 1010 °C and a 2 μm thick n-GaN/GaN was grown (carrier concentration -2.48 × 10 18 cm -3 ) [26] using H 2 . Silane flow rate for Si-GaN layer was maintained at 40 standard cubic centimeters per minute (SCCM) with a V/III ratio of 614. The InGaN/ GaN MQW (one, three and five periods) structures comprising of InGaN wells were grown at 720 °C with V/III ratio 11,625 (TMIn flow-13 μmol/min & TEGa flow rate -6.2 μmol/min) and the GaN barriers at 820 °C. Finally, Cp 2 Mg flow rate for Mg-GaN layer was 300 SCCM grown at 920 °C, continue to In-situ annealing at 700 °C for 15 min in all samples. Schematic view of the structures has been shown in the Fig. 1.

The crystalline quality, composition and thickness of the InGaN/ GaN MQW structures were determined using High resolution X-ray Diffractometer (HRXRD-PANalytical X'Pert Pro MRD). The morphology of InGaN/GaN MQW and the interfaces were performed using a High resolution transmission electron microscopy (HRTEM) (Hitachi, H-9000-NAR, LaB 6 filament, 300 kV, point to point resolution: 0.18 nm). Cross-section TEM samples were prepared by tripod polishing and subsequently polished with Ar + ion using Gatan precision ion polishing system (PIPS).The surface properties and roughness were analyzed by scanning electron microscopy (SEM, Carl Zeiss EVO18), atomic force microscopy (AFM, Park XE-100) respectively. Photoluminescence (PL, Spectra Physics) of the samples were measured at room temperature by 244 nm Ar + laser. The electrical properties of the samples were measured using hall measurement system (ECOPIA, HMS 5000).

Results and discussion

Structural analysis

Fig. 2 illustrates the ω-2θ scan of MQW structures were observed using HRXRD. Thicknesses and In compositions of the InGaN MQWs were obtained from the symmetric scans of (0 0 0 2) plane. Using the epitaxy smooth fit software (Fig. 2(III)), the InGaN well and GaN barrier thickness were determined as 3 ± 0.5 nm and 18 ± 0.5 nm respectively. The In compositions were estimated around ~16-18%. From the obtained HRXRD results, the high intense peaks manifests that, the grown n-GaN/GaN is oriented along (0 0 0 2) plane. The satellite peaks observed illustrates good periodicity in the MQW layers (Fig. 2(II)). It is worth to note that, the absence of satellite peaks in one period InGaN/ GaN QW (Fig. 2(I)), emphasize the non-uniformity in the InGaN/GaN interface which is evident from its surface roughness [START_REF] Sun | Short-wavelength light beam in situ monitoring growth of InGaN/GaN green LEDs by MOCVD[END_REF]. The full width at half maximum (FWHM) broadening of three periods InGaN/ GaN MQWs improved the crystalline randomization by exposing less interface roughness between InGaN and GaN layers in comparison to one period QW structure [START_REF] Nee | Characterization of Berthelot-type behaviors of InGaN/GaN semiconductor heterosystems[END_REF]. Also, the satellite peak narrowing of five periods InGaN/GaN MQW revealed the interface is atomically flat when compared with the other two samples (Fig. 2(II)). Among the three samples, the InGaN/GaN MQW structures grown at five periods exhibits better crystallinity [START_REF] Ren | Enhanced performance of InGaN/GaN based solar cells with an In 0.05 Ga 0.95 N ultra-thin inserting layer between GaN barrier and In 0.2 Ga 0.8 N well[END_REF].

The rocking-curve analysis in respect of GaN was carried out for all the samples. The FWHM of (0 0 0 2) plane rocking curve of GaN are estimated as 400, 370 and 372 arc sec, whereas for (1 0-1 5) it is 242, 230 and 246 arc sec. The TD density was calculated from the below equations [START_REF] Lazarev | High resolution synchrotron X-ray studies of phase separation phenomena and the scaling law for the threading dislocation densities reduction in high quality AlGaN heterostructures[END_REF],

= = D β b D α b 4.35 | | 4.35 | | edge edge Screw screw 2 2 2 2 (1)
where D edge is the edge dislocation density, D screw is the screw dislocation density and α, β refers to FWHM value measured from rocking curve diffraction peaks of (0 0 0 2) and (1 0-1 5) planes respectively.

Here, lattice constant a-axis (0.31878 nm) is the length of the burger vector for the edge dislocation density and c-axis (0.5185 nm) is the length of the burger vector of the screw dislocation density [START_REF] Choi | Analysis of the residual stress distribution in AlGaN/GaN high electron mobility transistors[END_REF]. The calculated TD density of GaN layers of the three samples are shown in Table 1. The TD density of GaN was found to be in the range from 6.32 × 10 8 to 5.99 × 10 8 cm -2 . The TD density in GaN base layer for three periods of InGaN/GaN MQW structure was lesser in comparison to the other two samples. This is attributed to the low tilt and twist disorientations with respect to the screw and edge dislocations originating from n-GaN/GaN layer. Fig. 3 shows the reciprocal space mapping (RSM) corresponding to the asymmetric (1 0-1 5) plane of InGaN/GaN MQW structures for one, three and five periods respectively. The high intensity GaN peak is observed on the intense contour lines in all the RSM studies and the InGaN/GaN MQW peaks are observed below the GaN peak. Using the epitaxy smooth fit software, the reciprocal lattice point (RLP) of Q x values was found to be 0.279046 rlu and 0.277604 rlu for the GaN buffer layer and corresponding peaks of three and five periods of InGaN/GaN MQWs respectively which indicates that the GaN layer, InGaN well and GaN barriers which accommodating on identical inplane lattice constants. As the critical thickness of the barrier and wells in the InGaN/GaN MQW structures did not exceed, the overlying InGaN MQWs are coherently strained with respect to the GaN base layer [START_REF] Farrell | High quantum efficiency InGaN/GaN multiple quantum well solar cells with spectral response extending out to 520 nm[END_REF]. This reveals that the InGaN/GaN MQWs are pseudomorphic to the GaN layer. The obtained results comply with the Fast Fourier Transformation (FFT) analysis.

Fig. 4 represents the cross-sectional HRTEM images of hexagonal GaN (ICSD#25676) layer and sapphire substrate (ICSD#9770) indexed using java electron microscopy software (JEMS) [START_REF] Stadelmann | JEMS java electron microscopy software[END_REF]. From figure, it is clear that, the interface between the layer and substrate is atomically flat. The SAED pattern signifies the epitaxial relationship of hexagonal GaN layer on sapphire substrate. The obtained SAED patterns complement the HRXRD results. The GaN/InGaN/GaN interface has exhibited surface morphology that is predominantly free from pits and stacking faults. The cross-sectional bright field TEM images Fig. 5(I,II) show high resolution image of InGaN/GaN MQW (three periods) which further displays the interfaces between the InGaN well (dark) and GaN (bright) barrier layer in InGaN/GaN MQW structures. The thickness of InGaN well and GaN barrier was estimated to be 3 nm and 20 nm respectively. Fig. 5(III) shows indexing of the FFT with both InGaN and GaN layers. The image shows continuous lattice fringes across the GaN/ InGaN/GaN interfaces, with no evidences of a misfit layer formation. FFT analysis confirms that the lattice plane alignments are similar in respect of both InGaN and GaN layers [START_REF] Ougazzaden | Epitaxial MOVPE Growth of Highly c-Axis Oriented InGaN/GaN Films on ZnO-Buffered Si (111) Substrates[END_REF]. The zone axis corresponding to GaN and InGaN layers were found similar to that of the growth direction.

Morphological analysis

Fig. 6 shows the sequence of AFM images comprising of InGaN/GaN MQW (one, three and five periods) structures respectively. The smooth and roughened surfaces of the p-GaN samples were clearly observed. These surface roughnesses of p-GaN can be attributed to TDs intersecting the active layer and the formation of Mg-H complexes in p-GaN layer [START_REF] Tsai | High efficiency and improved ESD characteristics of GaN-based LEDs with naturally textured surface grown by MOCVD[END_REF]. The roughness of the p-GaN surface decreases the reflection at the p-GaN/air interface which in turn increases the path light through the active region thereby increasing the light absorption of these structures. Rough p-GaN surface usually grows at low temperature, leading to a high growth rate that result in the formation of V-pits [START_REF] Farrell | High quantum efficiency InGaN/GaN multiple quantum well solar cells with spectral response extending out to 520 nm[END_REF]. As represented by the 2 μm 2 scans of AFM images, the p-GaN surface was characterized by spiral growth along with self-organized In (Ga)N like nanostructures and shallow type V-pits for one and three periods of InGaN/GaN MQW structures. The five periods of InGaN/GaN MQW structures showed step flow growth besides suppressing the nanostructures on the top of p-GaN layer. The root-mean-squared (RMS) roughness as 0.52 nm, 0.26 nm and 0.145 nm were observed for one, three and five periods respectively. In contrary, the 5 μm 2 AFM image for five periods showed significantly rough p-GaN surface with deep Vpits with considerably large RMS roughness of 29 nm. Fig. 7(I) shows the line profile with the pit around 1-2 μm. The observed V-pits were initiated on the TD intersection along the InGaN/GaN QW above the GaN layer (TD density ~10 8 cm -2 ). The depth of the pit was estimated to be 90-100 nm.

From the Fig. 7(II), the smaller and larger V-pits were observed from the SEM images for InGaN/GaN MQWs (five periods) samples. The Vpit density was calculated and found to be in the order of 0.16 × 10 8 cm -2 (Fig. 7(II)). The smaller V-pits were due to the edge TDs and larger V-pits compounds to screw or mixed TDs [START_REF] Chen | AFM application in IIInitride materials and devices[END_REF]. This emphasizes that the densities of the V-pits corroborates to the results calculated (TD density) from HRXRD.

Optical analysis

Room temperature PL spectra for the InGaN/GaN MQW structures for different periods were shown in Fig. 8. Near band edge emission (NBE) occurs at 363 nm for the n-GaN/GaN layer in all samples. The emissions of InGaN/GaN MQW structure were observed at 479 nm, 480 nm and 505 nm for one, three and five periods respectively. These peak emissions were found to be red shifted in the InGaN/GaN MQW structures which could be attributed to the QCSE resulting of the modification in polarization effect [START_REF] Ng | Structural and optical characterization of InGaN/GaN multiple quantum wells grown by molecular beam epitaxy[END_REF][START_REF] Liu | Energy band tilt in ultra-thin InGaN film affected by the surface adsorption and desorption[END_REF]. The PL peaks at 479 nm and 480 nm wavelength displays poor emission intensity caused by self-organized In(Ga)N like nanostructures. It is worth to note that, the emission at around 383 nm (3.2 eV) is usually occurs for highly Mgdoped GaN that is attributed to the transition between the Mg acceptor (Mg Ga ) and deep compensating donor of Mg Ga -V N complex. However, this does not clearly emerge from the PL spectra due to the lower Mg incorporation and partially reduced nitrogen concentration in one and three periods of the InGaN/GaN MQW structures respectively [START_REF] Chen | Enhanced Mg doping efficiency in p-type GaN by indium-surfactant-assisted delta doping method[END_REF].

The high intense green luminescence peak observed at 505 nm was may be due to the dislocations and pits on the surface of five period's sample. The Mg and related transitions shift to 405 nm (3 eV) in the Mg doped GaN layers for five period of InGaN/GaN MQW structures, implying enhancement in the Mg incorporation. This could be attributed to the decrease in the self-compensation effect of native donors and Mg atoms [START_REF] Kaufmann | Nature of the 2.8 eV photoluminescence band in Mg doped GaN[END_REF]. One could understand from the above result that the intensity of the red (660 nm) luminescence is an effect of the strong

Electrical analysis

The mobility, carrier concentration and resistivity of the low temperature p-GaN with a Cp 2 Mg flow rate of 300 SCCM in respect of the number of periods of the InGaN/GaN MQW structures are shown in Table 2. The carrier concentration increases from 2.26 × 10 18 to 2.43 × 10 18 cm -3 for one and three periods of InGaN/GaN MQW structures. However, the carrier concentration decreases for the five periods of the InGaN/GaN MQW structures. The carrier concentration ~10 18 cm -3 range point towards the Mg doped GaN related band around 3-3.2 eV [START_REF] Obloh | Self-compensation in Mg doped p-type GaN grown by MOCVD[END_REF]. The mobility of the samples were found to decrease gradually as 290, 275, 272 cm 2 /V s for one, three and five periods of InGaN/GaN MQW structures respectively. In the present case, the mobility of low temperature Mg doped GaN was higher when compared to the high temperature p-GaN. Nevertheless, this condition is only applicable for the Cp 2 Mg flow rate is less than 500 SCCM [START_REF] Ju | Effects of p-GaN growth temperature on a green InGaN/GaN multiple quantum well[END_REF]. As summarized in Table 2, high resistive nature of five periods in InGaN/GaN MQW structures was observed in comparison to the other two samples (one and three periods). Additionally, it revealed the semiconducting behavior for the all samples.

Conclusion

In the present study, InGaN/GaN MQW structures were grown by MOCVD on sapphire substrates. The thickness, composition of InGaN well and GaN barriers have been estimated using HRXRD. The InGaN and GaN thicknesses are correlated by cross sectional HR-TEM. FFT analysis demonstrated the GaN/InGaN/GaN lattice plane alignments along the growth direction. From the asymmetric (1 0-1 5) plane RSMs, it was found that the InGaN/GaN MQWs were grown as a coherently strained on the underlying n-GaN/GaN layer. The results indicate that five periods of InGaN/GaN MQWs has better surface interface and crystalline quality. PL study revealed the high intensity green emission in InGaN/GaN MQW (five periods) structures. From AFM results, it was observed that the width and depth of the V pits increases with increasing of InGaN/GaN MQWs periods. Also step flow growth initiations in the top of p-GaN layer on InGaN/GaN MQWs (five periods) were revealed. This can be attributed to the suppression of self-organized In(Ga)N like nanostructures and spiral growth mechanism. Hall measurement results revealed the semiconductor behavior as an effect of increasing the number of the periods in InGaN/GaN MQW structures. This work helps us to understand the growth of InGaN/GaN MQWs structures and its structural, morphological, optical and electrical properties. It can be suitable for the fabrication of high efficiency optoelectronic devices.

Fig. 1 .

 1 Fig. 1. Schematic diagram of the InGaN/GaN MQW structures.

Fig. 2 .

 2 Fig. 2. HRXRD (0 0 0 2) plane of (I) InGaN/GaN QW (one period) structure. (II) InGaN/GaN MQW (three and five periods) structure (III) InGaN/GaN MQW (five periods) for experimental (blue line) along with simulation (red line).

Fig. 3 .

 3 Fig. 3. RSM of the InGaN/GaN MQW structures (one, three, and five periods) along (1 0-1 5) plane.

Fig. 4 .

 4 Fig. 4. (I) Interface of n-GaN/GaN/Sapphire (II) Corresponding selected area electronic diffractions (SAED) pattern and (III) simulation with JEMS software.

Fig. 5 .Fig. 6 .

 56 Fig. 5. (I) GaN/InGaN/GaN interfaces representing InGaN/GaN MQW structures (three periods) (II) High resolution TEM of GaN/InGaN/GaN interfaces in three periods of InGaN/GaN MQW structures and corresponding FFT (inset view) (III) Simulation of JEMS software in InGaN and GaN.

7 .

 7 (I) Line profile obtained from AFM images for five periods (II) SEM images corresponding to (I).

Fig. 8 .

 8 Fig. 8. Photoluminescence spectra of InGaN/GaN MQW structures for different periods.

Table 1

 1 Summary of GaN dislocation densities in the InGaN/GaN MQW structures.

		Screw dislocations	Edge dislocations	TD (10 8 cm -2 )
		(10 8 cm -2 )	(10 8 cm -2 )
	1 Period	3.21	3.11	6.32
	3 Periods	2.75	2.81	5.56
	5 Periods	2.78	3.21	5.99

Samples

GaN dislocations in the InGaN/GaN MQW structures

Table 2

 2 Electrical properties of the InGaN/GaN MQW structures for different periods.

	Samples	Mobility	Carrier concentration	Resistivity
		cm 2 /V s	cm -3	ohm-cm
		RT	RT	RT
	1 Period	290	2.26 × 10 18	0.0095
	3 Periods	275	2.43 × 10 18	0.0093
	5 Periods	270	2.30 × 10 18	0.0099

Acknowledgement

The authors gratefully to acknowledge Department of Science and Technology (DST/TM/SERI/2K12/71(G)), Government of India for funding the research project.