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Exosomal Consignment in Renal Allograft Injury

Abstract

Exosomes are small mobile endocytic vesicles (30-120nm), shredded by every cell to conduct trafficking of cell generated cargo. They are found in almost all body fluids (blood, csf, saliva, urine). These include proteins, lipids, DNA, mi(cro)RNAs etc. In multicellular organisms, they are packaged into numerous vesicles mainly in exosomes to conduct their transport for various cellular activities which can be exploited clinically. Presently the survival of renal allograft is monitored by mostly invasive methods (tissue biopsy, Creatinine, GFR) where curving the injury is quite difficult. Hence potency of molecular markers like proteins and then circulating miRNAs came to picture for early detection of renal injury (Acute Kidney Injury-AKI and Chronic Kidney Disease-CKD). However, due to lack of specificity of circulating miRNAs lose their feasibility and the discovery of these exosomal cargos in cellular communication has become an efficient tool for treatment of various complicated clinical condition including renal allograft injury.

Keywords: micro RNAs,exosome, Renal Allograft Injury

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Introduction

Exosomal world: a prologue

Exosomes are membrane bound mobile vehicles that are found in almost all circulating body fluids like- blood, CSF, saliva, urine, etc. These are responsible for transport of respective cellular cargo to extracellular target sites [1]. Recent studies with exosomes have revealed that exosomal cargo delivery has many important biological, physiological and pathological significance thus, can be an effective diagnostic tool for various diseases [2]. Exosomes are small circulating units of 30-120 nm in diameter, generating from late endosomal compartments of cells by its cell membrane invagination or budding or released as shedding vesicles. Cellular cargos include proteins, lipids, DNAs, mRNAs, miRNAs, etc [1]. The exosomal cell membrane mainly constitute a limiting lipid bilayer, transmembrane proteins and a hydrophilic core containing proteins, mRNAs and microRNAs (miRNAs).

Exsosomes were first discovered by Pan and Johnstone in 1983 [3] when they found that the release of transferrin receptors into extracellular space during sheep reticulocyte maturation was released inside a type of small vesicles. In 1989 Johnstone regarded these mammalian cargo delivering vesicles as exosomes [1-5]. Valadi et al. in 2007 first described about the composition of exosomes that apart from proteins and lipids these also contains DNAs and RNAs [6] which are recorded in ExoCarta database [7,8] . The exosomal cargo delivery requires stimulation of target cell which may be direct by receptor mediated interactions or may aid in transport from cell of origin to various bioactive molecules e.g. membrane receptors, proteins, lipids, mRNAs, miRNAs, etc [7]. When exosomes deliver its contents into the respective target sites the property and behavior of these cells changes to a great extent [8]. It is also understood from various studies done in last couple of years that miRNA composition of parent cell and exosomal components vary a lot [8] and of all the components, miRNAs have drawn the attention due to their regulatory role in gene expression as these are protected against RNAase-dependent degradation [1-8]. Thus exosomal cell-to-cell communication influence both physiological as well as pathological environment of the body. These play important roles in immune reactions, tumorigenesis and in neurodegenerative disorders [1]. e.g. in prostate cancer, ovarian cancers, lymphoma glioblastoma, etc [1].

Biogenesis

Exosomes are formed from late endosomal compartments of cells through endosomal sorting complex required for transport (ESCRT-that recognizes ubiquitylated proteins) to deliver the cargo to target cell or to fuse with lysosomes to degrade the undesired contents [1]. Earlier these exosomes were only considered to be “garbage bags” as their diversified capabilities were unknown then. But now these are the most emerging field of research. The way of formation and secretion of these vesicles from mutlivesicular bodies (MVBs) are of two types [9]:

Microvesicles, which are directly shed from cell membrane.

Exosomes, which are released by exocytosis when MVBs fuse with plasma membrane.

Exosomes can be identified by transmission microscopy as a cup-shaped morphology with negative staining [10-12]. These can be concentrated in 1.10-1.21 g/ml section of a sucrose density gradient [10-12]. Exosomes can be identified by various protein markers e.g. tetraspanin proteins- CD63, CD9, CD81, HSP70 and HSP90, etc [1, 8]. ExoQuick (a one-step precipitation procedure for exosome extraction), Immuno affinity capture, Immunobead (EpCAM), combination of EpCAM and ultracentrifugation, size exclusion chromatography and EpCAM and followed by Quantitative PCR, Microarray techniques for extraction and quantification of exosomes [1,8,13].

Exosomes formation and secretion requires enzymes and ATP. First the cell membrane is internalized to produce endosomes. Subsequently, many small vesicles are formed within endosomes by invagination of its cell membranes [8, 14]. Such endosomes are called MVBs. Finally, the MVBs fuse with endosomal cell membranes to release intraluminal vesicles into extracellular space which become exosomes [14].

The secretion or cell-to-cell communication of exosomes requires certain regulatory factors which were first identified by Ostrowski et al. who observed that Rab27a and Rab27b were associated with exosomal secretion [8]. It was also found that effectors of Rab27- SYTL4 and EXPH5 could also inhibit exosomal secretion in HeLa cells [15]. Also Yu et al. discovered that tumor repressor protein p53 and its downstream effector TSAP6 were required for influencing exosome secretion [16]. Another working group, Baietti et al. observed the importance of syndecan-syntenin which directly interact with ALIX protein via Leu-Tyr-Pro-X(n)-Leu motif in secrection of exosomes by endosomal budding [17]. Koumangoye et al. found that disruption of lipid rafts in exosomal membranes could inhibit its internalization in breast cell carcinoma cell line [18]. Trafficking of exosomes to target sites occurs in following mechanisms:

The transmembrane proteins of exosomes directly interact with signaling receptors of target cell membranes [19].

The exosomal fusion with plasma membrane of recipient cells to deliver the cargo into their cytosol [20].

The exosomes internalization into recipient cells have two fates[21].

in one, some exosomes are engulfed by the cell and may merge with the cell’s endosome and undergo transcytosis

in other case, engulfed exosomes fuse with endosomes and mature into lysosomes for degradation.

As per ExoCarta database records, of all the components proteins and miRNAs have been exploited for various research to correlate some application with different diseased state that could render some remedy. Due to the regulatory role of miRNAs in gene expression these are used as recent area of research as diagnostic tool [8,22]. Goldie et al. demonstrated that among small RNAs, the percentage of miRNAs is higher in exosomes than in parent cells [23]. Studies done with exosomalmiRNAs shows there are specific sorting mechanisms for miRNAs into exosomes. These are:

The neural sphingomyelinase 2 (nSMase-2)-dependent pathway by Kosaka et al. [24].

The miRNA motif and sumoylated heterogeneous nuclear ribonucleoproteins (hnRNPs)-dependent pathway by Villarroya- Beltri et al. [25].

The 3’ end of the miRNA sequence-dependent pathway by Koppers-Lalic et al. [26].

The miRNA induced silencing complex (miRISC)-related pathway and human AGO2 protein [27].

In short there are specific sequence in miRNAs as well as enzymes and proteins that guide them for their sorting into exosomes [8]. Exosomes are shed by cells during both normal as well as pathological conditions. Thus several studies have been made with exosomes in diseased states.

A brief sketch on therapeutic exosomal cargos:

Exosomal miRNA: miRNAs are the recent findings in the field of clinical research which are thought to be crucial in depicting human health and diseases. These biomarkers can also be an indicator for rejection onset of transplanted allograft. miRNAs are a class of small 18-25 nucleotide (nt) long endogenous, noncoding RNAs which play an important role in regulating gene expression [28,29]. A single miRNA has the ability to regulate expression (mostly silencing) of hundreds of mRNAs and have been known to play important role in many cellular functions that include induction of post-translational repression, mRNA degradation/silencing and transcriptional inhibition, cell development, differentiation, proliferation and functional regulation of immune response among others [28-31].

The mystery behind the functional maturation of miRNAs has been solved by research in last couple of years. Similar to mRNAs, miRNAs are also initially transcribed in the nucleus [32]. miRNAs are at first transcribed in nucleus as primary transcript by RNA polymerase II called pri-miRNA [32-35]. This pri-miRNA has a hairpin stem-loop structure (~80nt length) that contains the mature miRNAs [36]. The pri-miRNA processing include recognition of the stem loop followed by its cleavage by DROSHA (a class 2 ribonuclease III) and DGCR8 (a miRNA-processing multiprotein complex) to release pre-miRNA [32-35]. Pre-miRNA is then recognized by Exportin-5 which allows its exports to cytosol for further maturation into 19-25 nucleotide strands by RNA endonuclease III called Dicer [32- 35, 37]. Cleavage of this pre-miRNA by Dicer result in double stranded (ds) RNA molecule of which one of the single strand with more unstable 5’ base pairing is selected and transferred to an Argonaute (AGO) protein and the other strand is degraded [35,38,39]. The selected strand induces silencing of mRNAs through RNA Induced Silencing Complex(RISC) thus affecting various cellular functions like cell differentiation, proliferation as well as development and functional regulation of immune system [32-35,40]. In normal tissues, RISC remain as a low molecular weight entity with reduced regulatory activity while under stressed or replicating conditions these become high molecular weight units with intensified regulatory activity when bound to mRNA [36]. Thus mRNA silencing by miRNAs results in lower protein levels in the body [36,41].

ExosomalProteins: Proteins are the building blocks of life in all living organisms. These are amino acid chains linked by peptide bonds. They are exquisite necessity in every cellular events, may it be the formation of new cells or cell repair. Thus, can be an important biomarker in depicting biological changes. Emerging research have exploited this idea and conducted various proteomic studies. A more burning concept is ofexosomal proteins. The work done and data obtained shows that besides miRNAs another important exosomal load isexosomal proteins. TrairakPisitkun et al had worked on urinary biomarkers and found that urinary exosomal proteins can also be an efficient protein biomarker in reporting renal tubulopathies and other renal disorders [42]. Exosomes normally found in urine accounts for around 3% of the total urinary protein contents and isolation of these exosomes can result in very large enrichment of urinary proteins derived from renal tubular epithelial cells [42]. The exosomal packaging occurs when the apical membrane proteins undergo endocytosis and packaged into MVBs. These MVBs undergo encapsulation of cytosolic proteins into small vesicles. Finally outer membrane of MVBs fuse with apical plasma membrane releasing exosomes containing both membrane and cytosolic proteins [42]. The proteomics study with LC-MS/MS coupled with upstream one dimensional SDS-PAGE separation experiments had disclosed a number of proteins associated with exosome biogenesis like class E vacuolar protein sorting (VPS), ALIX, Aquaporin 1, Aquaporin 2, ESCRT, etc [43]. A total of 295 proteins of urinary exosomeswere found to be associated with renal diseases and hypertension. These have been enlisted in Urinary Exosome Protein Database [42]. In another experiment where polypeptides were considered reflect that β2- microglobulin could be an indicator of damage of renal proximal tubule cells [42,44]. The techniques used to evaluate exosomal protein change is carried out by two dimensional difference in gel electrophoresis and change proteins are identified by mass spectroscopy and validated by Western Blotting [45]. Zhou et al worked with Fetuin-A, a protein of liver as an important exosomal protein that can indicate occurrence of AKI(Acute Kidney Injury) [45].

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Early Molecular Biomarkers for Renal allograft status

Years of research with renal allograft injury for either Acute Kidney Injury (AKI) or Chronic Kidney Disease (CKD) suggest that instead of invasive detection of allograft status there are scopes for early and non-invasive detection of injury through molecular markers. The studies made at the molecular level have disclosed the fact that acute and chronic rejections to a transplanted graft at preliminary stage can be ascertained by alteration in levels as well as expressions of various molecular markers involved in signaling of graft injury. These can be measured from blood/urine samples of patients. In acute rejection the early pathological change is defined by Ischemia-Reperfusion Injury (IRI) where altered expression of various miRNAs [46] is observed 3-7 days post-injury [47]. At later stage when rejection is in progress changes in levels of miR-210,-10a and -10b as well as some proteins (like perforin, granzyme A andB mRNA, FAS Ligand, FOXP3, etc) are observed [48]. Chronic rejection in early graft injury is generally associated with Interstitial Fibrosis and Tubular Atrophy (IF/TA). Pathophysiology of IF/ TA is the deposition of Extracellular matrix (ECM) proteins and Epithelial-Mesenchymal Transition (EMT) which can be stimulated by Transforming Growth Factor beta (TGF-β)/Smad signaling cascades. Ample of literature suggest that TGF-β/ Smad signaling can cause up-regulation and down-regulation of various miRNAs (miR-21,-192 & miR-29 and -200 families under IF/TA conditions) [49,50]. Even though these biomarkers have provided fruitful information but they lack specificity and their cellular source is unknown since they circulate. So to get a much clearer picture of a particular injured cell research at molecular level have unrevealed the next generation biomarker –exosomalmiRNAs for early, specific and non-invasive detection. Moreover their cellular source is also defined so they can deliver exact status of a particular cell [1,8].

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Urinary Exosomal proteins and miRNAs in renal allograft injury as Next Gen Molecular Biomarkers

Studies done with renal diseases is pretty less and still a burning area of research that reveals the fact that urinary exosomal proteins as well as miRNAs can be a potential therapeutic tool for kidney and associated diseases [1,8].

The urinary exosomal proteins can be easily attainable by noninvasive means for diagnosis of ESRD as well as Urinary Tract Infection (UTI) [1]. In 2006 Zhou et al. reported that urinary exosomal protein- fetuin A was found to be increased in AKI (Acute Kidney Injury) patients in ICU than AKI patients in normal care [1,41,45]. In 2008, same group discovered that activating transcription factor-3 (ATF-3) was found in exosomes isolated from AKI patients than CKD patients or control [41,45,51]. Thus suggesting urinary exosomal proteins could be a diagnostic tool. Moreover, a reduced level of urinary exosomal aquaporin-1 has been observed in ischemia-reperfusion injury in rats [7]. ExosomalmiRNAs demonstrate potential diagnostic biomarker for renal fibrosis [8]. MiR-29c and CD2APmRNA [52,53] were observed in urinary exosomes of renal fibrosis patients. The findings by Stefano Gatti1 et al. showed that bone marrow derived Mesenchymal Stem Cells (MSC) Microvesicles (MV) when administered immediately after IR injury can prevent AKI and CKD in rats [8,54] through their paracrine actions. Tara K Sigdel et al have described that in AKI patients with perturbation exosomal proteins like CLCA1, PROS1, KIAA0753 were observed. In addition to that exosomal ApoM is more than soluble ApoM [55]. M.W. Welker group found that in patients with chronic Hepatitis C serum soluble exosomal CD 81, a surface protein marker is elevated [56]..

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Future Prospective and limitations

Lots of work have been done with circulating miRNAs but due to their less specificity with the exact status of injured tissues and accuracy in determining role of a miRNA and its cellular source, still more feasible molecular markers have been searched and scientists have found that the circulating vehicles of cells-circulating exosomes that carry respective cellular cargo to the target sites to conduct cell-to-cell communication can be an option. These can be more proficient in delivering the most specific information on the status of any cell, may it is normal or injured cells. The molecular composition of exosomes that has been found till date is being recorded in the ExoCarta database. By exploiting these data in different pathological diseases scientists have done lots of research with carcinomas. In renal diseases also these exosomal miRNAs are being used to find out a means for noninvasive early detection of graft rejection. The conclusion drawn from these studies that proteins like fetuin-A and activating transcription factor-3 (ATF-3) can be used as marker in acute kidney disease and miR-29c and CD2AP mRNA are identified from urinary exosomes in renal fibrosis patients.

Thus, the various convergent studies made in the field of transplantation have led to the discovery of potential therapeutic targets- non-invasive urinary exosomal miRNAs and proteins which can be used to investigate and confirm the injury of transplanted allograft at an early stage. Though the data obtained define exosomes as an appropriate marker when compared with mRNAs, still it has few limitations:

Diverse isolation procedures that can affect exosomal contents,

Exosomes containing large number of miRNAs that affect the signaling of the cell together but not itself alone and

TDifficultly in measuring the exact quantity of a particular miRNAs in a exosome when miRNA is in low concentration.

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Conclusion

The exosome cell-to-cell communication mechanisms’ experiments are still at its infant stage. There is the need for development of more sophisticated techniques to detect the exact amount of specific functional exosomal proteins and miRNAs and their proper signaling pathways. Thus more investigation are still required to exploit exosomes in clinical fields as therapeutic targets.

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Markers for the Characterization of Liver Mesenchymal Stem Cell Mesenchymal stem cells (MSCs) show promise for use in regenerative medicine and tissue engineering. Bone marrow mesenchymal stem cells (BMSCs) are considered to be the standard, but MSCs can be isolated from many sources. Liver mesenchymal stem cells (LMSCs) are a promising alternative to BMSCs. One challenge in MSC research is immunophenotypic characterization of the cells, since there is no specific marker for MSCs to date; in addition, markers may differ among sources and among species. In this review, we will discuss the markers that have been tested on LMSCs from different species. Keywords: Liver; Mesenchymal Stem Cells; Characterization; Bone Marrow; Human; Animals Go to Introduction Mesenchymal stem cells (MSCs) have the characteristics of self-renewal, immune regulation and multipotency. Due to their multi-lineage differentiation potential [1], MSCs can be induced to differentiate into adipocytes, osteoblasts, chondrocytes, muscle cells, nerve cells, liver cells and pancreatic beta cells in vivo and in vitro. These characteristics make MSCs promising candidates for use in cell therapy and regenerative medicine [2-4]. MSCs were first described as stromal stem cells from the bone marrow that have a spindle shape in culture. However, although the bone marrow is considered the standard source for MSCs, MSCs isolated from other sources have different features in terms of surface markers, proliferation rates, and differentiation capability [5-7]. MSCs have been isolated from a number of other sources, such as adipose tissue, dental pulp, umbilical cord blood, and amniotic fluid [8-10]. One alternative source for MSCs is liver tissue. Scientists have isolated liver mesenchymal stem cells (LMSCs) from different species and attempted to characterize them in hopes that these cells could be a better alternative to bone marrow mesenchymal stem cells (BMSCs), especially for liver diseases [8]. One of the challenging aspects concerning MSCs in general is their characterization. In this mini-review, we will focus on studies that characterized LMSCs from different species, such as humans, mice, rats, rabbits, sheep, chickens and cattle. Human LMSCs and human BMSCs The International Society of Cell Therapy (ISCT) states that cultured human BMSCs are positive for expression of CD105, CD73, and CD90 and negative for expression of CD11b or CD14, CD19 or CD79a, CD34, CD45, and HLA-DR (11). CD105 is TGF-beta receptor III, which plays a role in TGF-beta signalling during differentiation into chondrocytes [12]. CD73 is known to be involved in MSC migration and to modulate adaptive immunity [13,14]. CD90 plays roles in mediating cell-cell interactions and leukocyte adhesion to endothelial cells [15,16]. Human LMSCs are also positive for expression of CD105, CD73 and CD90 Table 1. However, Najimi et al. [17] reported that LMSCs have low expression of CD105, and most other studies reported that LMSCs are positive for CD105. Additionally, LMSCs are positive for expression of CD44, CD29, CD166, and CD146, which is consistent with BMSC expression patterns [18-20]. LMSCs and BMSCs from other species In mice, LMSCs and BMSCs have nearly identical markers since both cell types are positive for CD44, CD29 CD105, CD49e, CD90 and Sca-1. However, mouse LMSCs are positive for CD73 [21], and mouse BMSCs have low expression of CD73 [22]. In rats, there are no studies on LMSCs; however, Payushina et al. [23] indicated that LMSCs are positive for expression of CD90.Jones et al. [24] reported that bovine BMSCs are positive for expression of CD166. In addition, bovine LMSCs are negative for expression of CD166 but positive for expression of CD44, CD29, CD73, CD90, and CD106 [25-43]. Click here to view Large Table 1 Symbols indicate marker expression levels: −: no expression; +/−: <5% expression; +: 5−50% expression, ++: 50−100% expression. Go to Conclusion Many markers have been tested on LMSCs, especially on human and mouse samples, and BMSCs and LMSCs have been reported to share most markers. However, additional studies are required to establish a standard panel of positive/negative markers for LMSCs, and studies are also required for further characterization of LMSCs from other species, especially species that are used as in vivo models for many diseases. Go to Data Availability The data used to support the findings of this study are available from the corresponding author upon request.

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Re-Examining the Genetic Bottleneck: Atavistic Regression in Acquired Traits Affects the Outcome for Many Subspecies at the Allelic Level- Juniper Publishers

Preface

Nodular or clear cell hidradenoma denotes an asymptomatic, exceptional, gradually progressive, benign, solid or cystic, intra-dermal adnexal neoplasm of sweat gland origin with eccrine or apocrine differentiation. Initially scripted by Mayer in 1941, the tumefaction describes with a dual subtype as hidradenoma with eccrine or period differentiation and hidradenoma with apocrine or clear cell differentiation [1]. Frequent betwixt fourth and eight-decade, peak incidence of the disorder is cogitated within sixth decade although clear cell hidradenoma can appear in the first decade. Of undetermined genesis, the cutaneous neoplasm arises from eccrine dermal adnexal glands although apocrine derivation is occasionally delineated. Clear cell hidradenoma or the nodular hidradenoma is a frequent histological subtype. The nomenclature includes captions such as eccrine acrospiroma, solid-cystic hidradenoma, clear cell acrospiroma, clear cell myoepithelioma, eccrine sweat gland adenoma, nodular hidradenoma, clear cell hidradenoma, cystic nodule hidradenoma, and eccrine acrospiroma.

Disease Characteristics

Eccrine acrospiroma elucidates as a solitary, firm, nodule with a preferable localization in the head and neck, face and upper extremities although lesions can be cogitated on the chest, shoulder, upper torso, upper or lower extremities. A female to male ratio of 1.7 :1 and a mean age of presentation at 37.2 years is exemplified. An estimated proportion of lesions are cogitated at the head (30.3%), upper limb (25.8%) and trunk (20.2%) [2,3]. Paediatric adnexal tumours demonstrate a predominantly follicular or apocrine/ eccrine genesis. Pilomatrixoma is a frequent and most encountered skin adnexal tumefaction situated in head and neck region. Tumours with sebaceous differentiation are distinctly infrequent. Hidradenoma is generally elucidated betwixt 20 to 50 years of age. Clear cell hidradenoma can occur as a polypoidal umbilical mass, congenital neck swelling or as axillary nodules in infants. Hidradenoma emerge as a gradually progressive, solitary, asymptomatic, firm, mobile tumefaction or nodule [3,4]. A few of the tumour nodules can demonstrate a serous effluvium or may ulcerate. No anatomical site is exempt from the appearance of the tumour. Lesions are disclosed in vulva in females, peri-anal region inmales, scalp, head and neck, face, lower eyelid, external auditory canal, knee and foot. The tumefaction can be associated with hyper-oestrogenaemia as oestrogen and progesterone receptors are discerned on the tumour cells. Hyper-oestrogenaemia can engender multitudinous lesions at various sites [4,5].

Clinical Elucidation

Tumefaction of hidradenoma appears as solitary or multiple lesions. A female preponderance is noted with implication of middle-aged individuals. Adult females can exhibit lesions confined to the vulva. Although the disorder is predominantly cogitated in adults, nodular hidradenoma can arise in children. The nodules are frequently asymptomatic, evolve gradually and rarely demonstrate clinical symptoms such as pain and serous discharge. Solid or cystic, well demarcated, asymptomatic, minimally progressive, endophytic, rarely exophytic nodules of varying magnitude are cogitated usually of dimension betwixt 0.5 centimetres to 3.0 centimetres. The tumefaction enunciates a minimal possibility of malignant transformation and probable ulceration. The generally solitary nodules are skin coloured or crimson and frequently elucidated on the scalp, face, thorax, abdomen and proximal extremities [5,6]. Unconventional clinical appearance is cogently described with tumours exceeding 3 centimetres in diameter, erosion of the superficial surface, a preponderant cystic component and aberrant locations such as the plantar region.

Histological Elucidation

Clear cell hidradenoma is a well circumscribed, un-encapsulated tumour as delineated on gross and morphological examination. Clear cell hidradenoma comprises of lobules of tumour cells situated in the dermis with extensions into subcutaneous fat. The tumefaction is segregated from the epidermis by a dormant Grenz zone. The neoplasm, characteristically confined to the upper dermis, demonstrates a congregation of dual population of miniscule or enlarged, monomorphic or polyhedral epithelial cells with copious clear or eosinophilic cytoplasm and miniature nuclei [6,7]. The dimorphic cell population elucidates polygonal cells with abundant glycogen thereby inducing a clear appearance to thecytoplasm admixed with elongated, darkly stained, miniature cells cogitated at the periphery.

Clear cell metamorphosis and/or squamous metaplasia can be a predominant feature within the tumour cell accumulates. Focal apocrine elements are evidently displayed. The tumour exhibits diverse composites and nodules of benign epithelial cells articulating miniscule ductules and duct lumina generally cogitated within the upper dermis. Duct like configurations recapitulate the eccrine ducts. Additionally, slit like spaces lined by concentric layer of squamous epithelial cells are exemplified. Numerous cystic spaces are elucidated on account of tumour cell degeneration. Apocrine secretion can be delineated within the ductular lumen due to decapitation of cytoplasmic vacuoles [7,8]. Squamous, sebaceous or mucinous epithelial differentiation can be enunciated in specific lesions. The cellular component is variable, although clear cell predominance emerges in proportionately one third instances. Distinctive lesions are enveloped in an intact dermal integument or can demonstrate focal and superficial ulceration with discharge of serous fluid. The tumefaction exhibits singularly solid or cystic elements or an admixture of solid and cystic components in varying proportions or a clear cell predominance. Solid areas enunciate epithelial lobules comprised of preponderant polyhedral cells with a basophilic cytoplasm and clear cells with glycogen containing cells with a clear cytoplasm. Clear cells contain glycogen or a diastase resistant substance which stains with periodic acid Schiff’s (PAS) reagent. Lipid vacuoles are not elucidated.

Clear cells denote a metabolic variant of epidermoid cells, rather than an aberrant category of tumour differentiation. Stroma intervening within the lobules enunciate delicate,vascularised cords of fibrous tissue or dense hyalinised tissue. Myxoid and chondroid stroma are infrequent. Exophytic growth with superficial surface erosion of the tumour is observed [8,9]. As the histological description and annotation is competent, confirmation with immune histochemistry is not a cogent, essential exercise. Malignant transformation is infrequent and is characterized by morphological parameters such as nuclear atypia, necrosis and atypical mitosis. A distinctive histological enunciation may not accurately predict the clinical behaviour of clear cell hidradenoma (Figure 1–14).

Distinguishing Diagnosis

Clear cell hidradenoma necessitates a distinction from a concordant lesion such as haemangioma, glomus tumour, cutaneous lymphoma, dermatofibrosarcoma protuberans, leiomyoma, follicular cyst, trichilemmoma or adjunctive sweat gland or adnexal tumours which are clinically indistinguishable [9,10]. Clear cell neoplasm confined to the dermis necessitate a segregation from metastatic malignancies and primary skin tumours with follicular, sebaceous and sweat gland differentiation. Clear cell hidradenoma in adults require a demarcation from metastatic renal cell carcinoma. However, nodules of hidradenoma are devoid of the classic pulsating vascular prominence as delineated in malignant deposits of renal cell carcinoma [10,11]. Although clear cell hidradenoma, also termed as eccrine acrospiroma, is predominantly a benign neoplasm, the nodules can be subjected to malignant transformation. Engendered malignancy is cogitated as “clear cell hidradenocarcinoma” or “malignant clear cell hidradenoma”. The malignant counterpart is an exceptional neoplasm and is characterized by an infiltrative tumour perimeter, cellular atypia and innumerable atypical mitosis [11,12].

Therapeutic Options

A comprehensive surgical extermination is a pre-requisite in managing clear cell hidradenoma as the nodules are endowed with an enhanced potential of tumour recurrence. Awide tumour perimeter on surgical excision is mandated for a detailed histological confirmation which ensures minimal probable reoccurrence and evaluating prospective malignant transformation. A surgical eradication of the neoplasm with a broad perimeter is the preferred therapy. Malignant transformation is infrequent although neoplasm such as clear cell hidradenocarcinoma tend to reappear. Tumefaction undergoing malignant conversion demonstrate an aggressive clinical course, disseminated disease with adjuvant, enhanced mortality [12,13].

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MazEF homologous Modules System and A Post-segregational killing Mechanism (Bacteriostatic & Bactericidal Mechanism) of Novel Compound Isolated from Spondias monbinon Escherichia Coli and Bacillus Subtilis- Juniper Publishers

Abstract

The basic objective of this research work is to evaluate the mechanism of action of compound Epigallocatechin, Epicatechin and Stigmasterol Phytosterol (Synergy), Aspidofractinine-3-methanol) and Terephthalic acid, dodecyl 2-ethylhexyl ester with Selected clinical isolates by using molecular biomarker MazEF9 TA system. Toxin-Antitoxin (TA) systems are highly conserved in members of the Gram positive +ve and Gram negative –ve bacteria which has been proposed to play an important role in physiology and virulence. Clinical microorganisms were cultured and Sub-culturing in Department of Microbiology and Centre for Biocomputing and Drug Development (CBDD), Adekunle Ajasin University, Akungba Akoko, Ondo-state, Nigeria. A 12 hours old culture of each microorganism was re-suspended in plant extract at 1000 μg mL in a total volume of 500μl for 0, 15, 30, 45, 60, and 180 minutes.

Keywords: Post segregational killing mechanism epigallocatechin; Epicatechin; Stigmasterol phytosterol; Mazef TA System; Bacteriostatic

Introduction

Spondiasmombin L. (Anacardiacaea) also known as hog plum is a plant that grows in almost every part of the world. It is fruit feriousdecidous plant of about 20m high that grows in the rain forest and the coastal area of Africa. It is known locally as “iyeye” by the Yoruba people of Nigeria Ripped fruits are eaten out hand by the old and young and processed into ice-cream, cool beverages, wine, jam and other preservatives. Spondiasmombin alsofound application in folk medicine. Infusion of its leaves has been used for a long time, without any report of collateral effect due to its anti- vitrotic activity against the herpes virus [1]. A tea of the flowers and the leaves is taken to relieve stomachache biliousness, urethritis, cystitis and eye and throat inflammation. Herbalist in South Western Nigeria use the plant in the treatment of typhoid, tuberculosis, diabetics, nervous disorders and psychiatric disorders. Offiah, Anyanwu [2] have reported the abortifacient activity of the aqueous leaf extract of Spondiamombin. In addition, the anthelmintic, molluscicidal, anxiolytic, anti-bacteria, antiviral effect of the plant have been previously described. Idu et al. [3] reported the inhibitory activity of Spondiamombin against Cycasrevoluta induced carcinogenesis. The discovery of toxin-antitoxin gene pairs (also called homologous modules) on extra-chromosomal elements of Escherichia coli, Bacillus subtilis and particularly the discovery of homologous modules on the bacterial chromosome, suggest that a potential for programmed cell death may be inherent in bacterial cultures which is under the scope of this research work, this was reported on the E. coli / Bacillus subtilis mazEF system, a regulatable addiction module located on the bacterial chromosome. MazF is a stable toxin and a labile antitoxin. it show that cell death mediated by the E. coli /Bacillus subtilis mazEF module can be triggered by several medicinal plant extract and antibiotics like Rifampicin, chloramphenicol, and spectinomycinetc, that are general inhibitors of transcription and translation. These medicinal plant extract and antibiotics alike inhibit the continuous expression of the labile antitoxin MazE, and as a result, the stable toxin MazF causes bacteria cell death. The results of this research have implications for the possible mode(s) of action of this group of medicinal plant and antibiotics on selected clinical isolates. The mode of action of the medicinal plants may be either bacteriostatic or bacteriocide [4] but the scope of this research work is based on bacteriostatic mechanism of action of isolated novel compound on selected clinical organisms (E. coli and B. subtilis).

In Escherichia coli/Bacillus subtilis cultures, programmed cell death is mediated through a unique genetic system. This system, called “homologous module,” consists of a pair of genes that specify for two components: a stable toxin and an unstable antitoxin which prevents the lethal action of the toxin. Until recently, such genetic systems for bacterial programmed cell death have been found mainly in E. coli and Bacillus subtilis on low-copy-number plasmids, where they are responsible for what is called the postsegregational killing mechanism. When bacteria lose the plasmid( s) (or other extra-chromosomal elements), the cured cells are selectively killed because the unstable antitoxin is degraded faster than is the more stable toxin [5]. Thus, the cells are “addicted homologous” to the short-lived product, since its de novo synthesis is essential for cell survival [6].Therefore, these homologous modules have been implicated as having a role in maintaining stability in the host of the extrachromosomal elements on which they are bounded, Jensen, Gerdes [7], once this stability is destroyed, this will inevitably leads to the death of the bacterial cell. Pairs of genes homologous to some of these extra-chromosomal addiction modules have been found on the E. coli and Bacillus subtilis chromosome Aizenman et al. [8], Gerdes et al. [9]. The mazEF module consists of two adjacent genes, mazE and mazF, located in the rel operon downstream from the relA gene Metzger et al. [10].

In the study, mazEF was found to have the properties required for an addiction module:

(i) MazF is toxic and MazE is antitoxic

(ii) MazF is long lived, while MazE is a labile protein degraded in vivo by the ATP-dependent ClpPA serine protease

(iii) MazE and MazF interact

(iv) MazE and MazF are coexpressed

Both MazF and MazE are expressed thereby leading overexpressed killing effects of bacteria cell, Moreover, the mazEF system has a unique property: its expression is inhibited by guanosine 3′,5′-bispyrophosphate (ppGpp), which is synthesized under conditions of extreme amino acid starvation by the RelA protein [11] thereby leading to the death of the bacteria cell. Based on these properties of mazEF and on the requirement for the continuous expression of MazE to program cell death, members of our group offered a model for programmed cell death under conditions of nutrient starvation [8]. In this study, MazF (toxic) is used to stimulate the programme cell death of the clinical isolates and evaluates the efficacy of novel compound isolated from Spondiasmombn plant on the selected clinical organisms.

Materials and Method

Microorganism for the research work

Clinical microorganisms were used for this research work, which comprised Escherichia coli ATCC 25922and Bacillus subtilis.

Sources of microorganisms

All typed strains used for this research work were purchased from the University of Pennsylvania, School of Medicine, Philadelphia, United States of America (USA), in an America Type Culture Collection (ATCC) , and the other locally isolated bacteria and fungi were clinical organisms collected from Central Medical Laboratory( CML), Obafemi Awolowo University Teaching, Hospital (OAUTH), Ile Ife, Osun State, and the Institute of Advance Medical Research and Training (IMRAT),University College Hospital, Ibadan, Oyo State Nigeria.

Authentication of test microorganisms

The identity of the test organisms was confirmed using Biomerieux, France, API 20E Kits for bacteria as specified by the manufacturer’s instruction. Analytical Profile Index [12].

Isolation of RNA.

A 12 hours old culture of each microorganism was re-suspended in plant extract at 1000μg mL in a total volume of 500μl for 0, 15, 30, 45, 60, and 180 minutes. The cells were pelleted by centrifugation at 5000g for 5 minutes. The pellets were rinsed twice in phosphate buffer saline (PBS). Then 1/10 volume of 95% ethanol plus 5% saturated phenol were added to the pellets to stabilise cellular RNA. The cells were then re-harvested by centrifugation (8200g, 4°C and 2 minutes). The supernatant was aspirated and pellets re-suspended in 800 μl of lysis buffer (10mMTris, adjusted to pH 8.0 with HCl, 1mM EDTA) and 8.3 U/ml Ready-LyseTM Lysozyme Solution. After the pellets were re-suspended, 80μl of a 10% SDS solution was added, mixed and incubated for 2 minutes at 64°C. Then 88μl of 1 M NaOAc (pH 5.2) was mixed with the lysate followed by an equal volume of water and saturated phenol was added. This was incubated at 64°C for 6 minutes while inverting the tubes every 40 seconds. The aqueous phase was separated following centrifugation at 21,000g for 10 minutes at 4 °C. The RNA was precipitated from the aqueous layer using 1/10 volume of 3M NaOAc (pH 5.2), 1mM EDTA and 2 volumes cold EtOH and centrifugation at 21,000g for 25 minutes at 4°C. Pellets were washed with ice cold 80% EtOH and centrifuged at 21,000g for 5 minutes at 4°C. The ethanol was carefully removed and the pellets were air dried for 20 minutes. The pellets from each split

Solation of RNA from bacterial cell

Figure 1.

PCR protocol

Reverse Transcription–PCR reaction was performed in a 15.0μl final volume. Briefly, 1μl template cDNA (~40 ng) was combined with 1.0μl of forward primer (5nM), 1.0μl of reverse primer(5nM), 4.5 ml nuclease-free water and 7.5 μl of Taq 2X Master Mix. Thermo cycling was performed by 40 cycles at 95°C for 15 seconds, 60 °C for 15 seconds and 72°C for 15 seconds. Analysis of the PCR products was performed using 1.5% agarose gel solution in TBE buffer and visualisation was enabled by soaking gel in ethidium bromide solution for 10 minutes and UV-transilluminator. The data obtained were analyed using Graph pad prism version 6.01 description and frequency. statistic was generated to describe the diameter of inhibition. quantitative phytochemical constituent and toxicological prameter to test for the level of significance [1].

Gel electrophoresis

Assessment of Polymerase Chain Reaction products (amplicons) were electrophoresed in 0.5% of agarose gel using 0.5×TBE buffer (2.6g of Tris base, 5g of Tris boric acid and 2 ml of 0.5M EDTA and adjusted to pH 8.3 with the sodium hydroxide pellet) with 0.5μ lethidum bromide. The expression product was visualized as bands by UV-transilluminator [1] Table 1.

Results

The MazEFhomologous modules system and a Post-segregational killing mechanism (Bacteriostatic & Bactericidal mechanism) of novel compound isolated from Spondiasmonbinon Escherichia coli and Bacillus subtilis were demonstrated in Figure 1–6. The novel compound isolate from Spondiasmombin include the following, (A3-Epigallocatechin, Epicatechin and Stigmasterol phytosterol (synergy), A3-Aspidofractinine-3-methanol) and F3(Terephthalic acid, dodecyl 2-ethylhexyl ester) and the gene expression MazF9was observed in Escherichia coli and bacillus subtilis. Figure 1 shows Bacillus subtilis, mazEF homologous modules system and Post-segregational killing mechanism (Bacteriostatic & Bactericidal mechanism) of compounds A1(Epigallocatechin, Epicatechin and Stigmasterol Phytosterol (Synergy), A3(Aspidofractinine- 3-methanol) and F3 (Terephthalic dodecyl 2-ethylhexyl ester) by MazEF9 (Toxin/ antitoxin sensor. In Figure 2,3 and 4. It was observed that compounds A1, A3 and F3 have a deleterious effect on the cell (via DNA) of the test organism. At 180 minutes, the death phase was ascertained. This is graphically demonstrated in the figure1. Both MazE (toxin) and MazF (antitoxin) were produced, which both were used to activate the death phase of the cell through Post-segregational killing mechanism (Bacteriostatic & Bactericidal mechanism) of MazEFhomologous modules system on E. coli and B. Subtilis. The compounds have bacteriostatic and bactericidal effect on the test organisms (E. coli and B. subtilis). Figure 5 shows E.coli, mazEF homologous modules system and Post-segregational killing mechanism (Bacteriostatic & Bactericidal mechanism)of A1(Epigallocatechin, Epicatechin and Stigmasterol Phytosterol (Synergy),A3 (Aspidofractinine-3-methanol) and F3 (Terephthalic dodecyl 2-ethylhexyl ester)by gene expression MazEF9 (Toxin/ antitoxin sensor ) bacteriostatic and bactericidal effect as the mechanism of action compounds of A1, A3 and F3 as observed in (Figure 6–8) respectively.

Discussion

The purpose of the research work is to determine the Post-segregational killing mechanism (Bacteriostatic & Bactericidal mechanism) of novel compound isolated from Spondiasmonbinon Escherichia coli and Bacillus subtilis by MazEFhomologous modules system. action of compound A1 (Epigallocatechin, Epicatechin and Stigmasterol Phytosterol (Synergy), A3(Aspidofractinine-3-methanol) and F3(Terephthalic acid, dodecyl 2-ethylhexyl ester) with two Selected microorganism (Gram positive +ve), (Gram negative –ve) were demonstrated by toxin-antitoxin gene pairs (also called addiction modules) on extra-chromosomal elements of Escherichia coli, Bacillus subtilis All figs describe the mechanism of action of the isolated compound of ethyl acetate stem bark extract of Spondiasmombin extract. Nine of these TA systems belong to the mazEF family, encoding the intracellular MazF toxin and its antitoxin. Another mechanism of action of compound A1, A3 and F3 on the selected microbe E. coli and Bacillus subtilis is by probing for the toxin-antitoxin Maz F9 bio sensor. It was observed that in Figure 1–3. A1 MazF9, A3Maz F9 and F3 MazF9 stimulate the production of toxin/antitoxin system in the cell of the microorganism, used for this study and a greater effect were found between 0 to 15 minutes and decrease in effect were found at 30 to 180 minutes, this effect of toxin-antitoxin leads to the death of the organism between 0 to 180 minutes, the death ratio is measured and cells were destroyed or damaged by stimulation of toxin-antitoxin system. Toxin-antitoxin system are induced to measure the death phase at 0–180 minutes and this study was conducted to investigate the role of MazF9 to isolated compound. It was also observed that over expression of MazF9 induced a state of irreversible bacteriostasis consistent with the results of the previous study [13- 15]. The main finding of this work is that E. coli mazEF-mediated cell death can be triggered by medicinal plant that are general inhibitors of transcription and/or translation. it was shown that these Spondiasmombin reduce the cellular level of the antitoxic labile protein MazE and seem thereby to permit the lethal action of the toxic protein MazF. The effect of the Spondiasmombin both on cell death and on the reduction in the cellular level of MazE [16].

The isolated compound has a great effect on selected organism. It was observed that the effect of gene MazF9 has more lethal effects in E. coli, taking a cognizant from figure 5–8 represented by A1MazF9, A3MazF9 and F3MazF9. It was observed that the compound complete inhibits the E. coli at 180 minutes i.e. the compound has a better activity on E. coli than B. subtilis. The reasons for this activity must be as a result of their cell wall, the Gram negative (E. coli) has a greater activity because of a thin composition of the peptidoglycan cell wall compares to the gram positive (B. subtilis) which has a large composition of peptidoglycan in the cell wall. The gram negative is easily affected by antibiotic compound and compares the gram positive with very thick cell wall, this account for the activity of MazF9 more on E. coli than B. subtilis but their death rate was constantly measured, and toxin-antitoxin activity were clearly depleted [17].

In analogy to the programmed cell death apparatus in eukaryotic cells [18] reported that the suicide machinery in bacterial cells is always present, it is only requires a trigger to activate it. Moreover, at least for E. coli, the straightforward choice is death caused by a stable intracellular toxin (in this case MazF). The choice of life over the “default” death requires a dynamic antagonistic process manifested either by the continued production of the unstable antitoxin (in this case MazE) or by a process that would prevent the degradation of the unstable antitoxin. Thus, cell death could be caused by anything that would prevent the continuous expression of the antitoxic protein MazE [4]. It should be noted that toxin/ antitoxin system is widespread in bacterial genomes and contribute to prokaryotic stress adaptation and the formation of persister cells biofilms [19]. The mechanism of toxin components is to exert their effects in different ways by targeting essential cellular functions such as DNA replication, protein synthesis, cell division, peptidoglycan biosynthesis (see above) and ribosome assembly.

However, RNA cleavage is the most prevalent mode of action in the pathway [20]. Vasperet al., [21]reported that in E.coli (see above), one of the most well characterized toxin-antoxin gene is MazF9, an operan that encodes that intracellular MazF and its cognate antitoxin MazF9 toxin cleaves MRNA and DNA at 3,5 or 7 base recognition sequences in different bacteria and have been implicated in controlling cellular responses to various adverse conditions encountered by the bacteria in the host. Other mechanism of MazF9 in E. coli is MazF9 protein cleaves both MRNA and 16s ribosomal RNA and proposed to generate a subpopulation of stress ribosomes, thereby enabling the translation of leaderless transcript [22]. MazF9 system in E. coli are activated on exposure to numerous stress condition in an extracellular death factor-dependent manner. Another mechanism of action of compound A1, A3 and F3 on the selected microbes E. coli and Bacillus subtilis was demonstrated by probing for the toxin-antitoxin MazF9 gene. This effect of toxin-antitoxin MazF9 biosensor leads to the death of the organisms between 0 to 180 minutes. The compounds have more lethal effects as they completely inhibited the E. coli at 180 minutes. In addition, the compounds have a better activity on E. coli than B. subtilis. The reasons for this activity must be as a result of differences in their cell wall compositions.

This results in Figure 1–8, showing that the mazEF system is responsible for approximately 90% killing by Spondiasmombin and other medicinal plant and antibiotics alike may illuminate, the until now elusive cause of E. coli killing by medicinal plant. Furthermore, medicinal plant like Spondiasmombin are considered to be bacteriostatic and bactericidal in action [23]. It should be noted that toxin system is widespread in bacterial genomes and contribute to prokaryotic stress adaptation and the formation of persister cells biofilms [19]. The mechanism of toxin components is to exert their effects in different ways by targeting essential cellular functions such as DNA replication, protein synthesis, cell division, peptidoglycan biosynthesis (see above) and ribosome assembly. The mechanism of action of toxin-antitoxin gene MazF9 include RNA cleavage [20]., MRNA and DNA cleavage and protein cleavage of both MRNA and 16s ribosomal RNA [22].

Conclusion

This is better method of measuring the mechanism of action of novel compound by triggering the toxin/antitoxin cell death of inherent nature of bacterial cell.

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