Shared Expression of Mucin12 contributein bothcommon antigenicity of host-parasite relationship between Ascaris Lumbricoides and Human Small Intestine

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species should be italicised.

Authors
Affiliations*Corresponding author:

Commented [SC ED2]: The title of the paper should beconcise and yet present the main research focus of thepaper. We have therefore, revised it to make it impressive
and informative for the readers.
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Conflict of interest:

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Please follow the below format for the names:First Author1, Second Author2*, Third Author3

If available, please provide the e-mail address of eachauthor.
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Commented [SC ED4]: Please provide full postal
address of all the affiliations of the authors you havementioned above.
An example of the format is given below:
1First Department, First University, Address, City, Country

Name
Commented [SE5]: Please provide the details ofcorresponding author from among the authors that you
have listed above. The corresponding author will be theone who will communicate with the journal after
submission, on behalf of remaining authors in the paper.
Commented [SE6]: Please provide a declaration of any
conflict of interest. If there are no conflict of interest to
declare, the authors can mention “There are no conflict ofinterests to declare.”SummaryAbstract

Ascaris lumbricoides is one of the most common parasites in the world. The

Commented [SC ED7]: The abstract reflects the aim of
the study and the methods implemented to achieve thepurpose of this research study is to focuses on the host specificity of human Ascaris.

study’s objectives. However, you may consider to provide

lumbricoides, which is a parasitic parasite ofin the small intestine and is also one of thea brief introductory statement about already known host-commonest parasites worldwide. As part of this investigation, we examined, at a thepathogen interaction mechanism that would help in

genetic level, we examined the common antigenicity existing in A. lumbricoides andconveying the focus of the study clearly.

human small intestinal mucosa to unravel the this host–parasite relationship. We

Formatted: Font: Not Italic, Not Highlightobtained three DNA clones after by screening analysis for common antigenicity of using

Commented [SC ED8]: This is a lengthy sentence and isa human colon cDNA library on common antigenicity usingand anti-A. lumbricoides

filled with jargons. Hence, we have edited it to make apolyclonal antibodiesy. After sequencing analysis, we identified one of them is theconcise yet clearer and meaningful context to yourtransmembrane mucin12 gene was identified as a gene of interest. SThe specific signals

research.

of immunoe-staining with polyclonal anti-mucin12 antibodiesy were observed in the
mucous secretory organs, epidermis, and intestinal canal of A. lumbricoides. These

Commented [SC ED9]: It would be ideal to mention thesignals were disappeared when immunohistochemistry was performed using pre-molecular weight of this glycoprotein and present aabsorbed polyclonal antibodiesy with a specific peptide. These results suggested thatmeaningful sentence.

mucin12mucin12 was is localized in the mucous secretory organs to in the epidermis of

A. lumbricoides. Furthermore, we examined the site of mucin12mucin12 localization onin the host side,; the specific mucin12 signals of mucin12 were observed on the mucousal
epithelial present around intestinal crypts and villi of the small intestine. Therefore, it
iswe suggested that mucin12mucin12 is one of thea proteins that show thes common
antigenicity in both parasites, A. lumbricoides and its host. It is presumed that adult A.

lumbricoides live in its their ideal preferred environment, which is the small intestine,by secreting mucin12mucin12 , which is the common antigenicity in the small intestine,
to avoid being attacked by the host immune system.
Keyw Words:
Human Ascaris Lumbricoides, Human human Small small Intestinal intestinal

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the Abstract by stating the defensive mechanism of theparasite in human small intestine, please note that this isquite a known fact reported in several papers. Hence, thefinal statements here should reflect the potential of yourfindings from a diagnostic and prognostic view to appeal
the reader’s attention.
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Commented [SC ED11]: We have added a relevant
keyword to enhance the paper’s chances of visibility andsearchability by readers online after it gets published.

Mucosamucosa, Mucin12mucin12,

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Hosthost–-parasite relationship, sequencing,

you are required to place a list of abbreviations in afootnote on the first page of the article.

Abbreviations

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1. Introduction
Infection with the parasite A. lumbricoides infection is a disease caused byparasitizing of A. lumbricoides and isis widespread throughout the world. Many Several
cases infections develop in tropical, subtropical, and temperate regions, although a few

confusing. Please note the changes we have done byCommented [SE14]: Tip: Serial comma
In American English, a comma (called serial or oxfordcomma) is inserted before “and” in a series.

...

also develop infew may also occur in cool regions. In Japan, the there weremanyprevalence of A. lumbricoides patients infection increased to such an extent afterWorld War II and that it was called deemed a national affliction;, but however, thenumber of patients prominently has decreased considerably, thanks to groupdisinfestation, usage of chemical fertilizers, and the improved conditions oflivingenvironment. However, iIncreased international travel ing has been causing new

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problems in recent years because infection sources have increased due toof infected

and the flow of research findings reported in unclear. We

travelers from overseas countries entering Japan.

have edited the sentence and added punctuations

Infection begins when mature Looking at the life history of A. lumbricoides , at firstmature eggs were are ingested orally ingested. Once they reach the , and reached to thesmall intestine, they and hatches., and Hatching the larvae invade the intestinal walls.
of the small intestine, andThey are then able to enter the systemic circulationthe
circulatory system via the portal vein and reach the lungs from the heart. They breakwherever applicable to enhance the overall readability.
Commented [QA16]: I have edited this sentence to bring
the focus of the reader to the ‘infection’ rather than the‘life history’ of the parasite.

rupture the alveoli and areis swallowed once more by through the pharynx via the,bronchus, and the trachea, and enabling them to returns to the small intestine again .
Finally, they become adultsreach maturity in the small intestine, where they matureand remain and stay. The human small intestine is considered to be theI ideal habitat

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for A. lumbricoides can be the human small intestine, but although its immunologicalof texts and hence we have edited it to present a concise

escape mechanism has not yet been sufficiently clearly elucidated yet. Few gene level

context.

studies of the genetics of A. lumbricoides exist, and no research has been conducted
regarding concerning the common antigenicity between A. lumbricoides and the humansmall intestine has been conducted.

Commented [SC ED18]: This is a well-known fact andhence, please provide a suitable reference to it.

However, some studies have investigated the biochemistry of the intestinal
mucosa and its relevance in A. lumbricoides infection. As for research concerning A.

lumbricoides and human intestinal mucosa, Scientists have proved reported theFormatted: Not Highlight

existence of an antibody against nematodes in the blood serum of ulcerative colitis
patients through using the Ouchterlony method.

The researcher reported the existenceof common antigen substances in the cortical laminae and basal laminae of the cuticles
on of A. lumbricoides side. Antigens common to A. lumbricoides,

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citation.

local existenceare existof common antigens with A. lumbricoides around the basement membrane in normal
human intestinal mucosa, and that a protein with a molecular weight of 41.38 kDa in a

Commented [QA20]: Do you want to depict any specificcrude antigen of normal intestinal mucosa antigen is the substance that owns shares asignificance of this molecular weight of protein in thecommon antigen with A. lumbricoides.

parasite as well as human small intestine mucosa?

2. Materials and mMethods
2.1 Preparation of A. lumbricoides crude antigen and polyclonal antibody

Commented [SC ED21]: This sentence should bereferenced for the author to refer to the existing findings
reported in similar context.

AThe adult warm of A. lumbricoides were homogenized and the crude antigen
was extracted using a homogenizer with PBS at 4°C℃. RThe rabbit polyclonal antibody
specific for to A. lumbricoides were was kindly donated from by Dr. Ishida [5].

Commented [SE22]: Please provide details regarding
the affiliation. E.g.,

2.2 Screening using against a human colon cDNA library
In order to obtainWe screened for the a cDNA clone that show determines the

Department/university/state/country.
Formatted: Not Highlightcommoncommon antigenicity between host and A. lumbricoides and its host , we used
the screening method ofusing a λ-phage method using and polyclonal anti - A.

Commented [SE23]: What is the company/clone of thelumbricoides antibodyies. Briefly, the plaques that were produced by using λ-phage

secondary antibody used for final detection?

human colon cDNA librariesy were transferred to the a nitrate cellulose membrane,
which was saturated with 10 mM IPTG. The membranes were screened with polyclonal

Commented [SC ED24]: The abbreviation should beanti-A. lumbricoides antibody antibodies and some of positive phage clones were

elaborated to help the reader understand the chemical

obtained identified by developing color using development using 3.3- diaminobenzidines-

composition of this reagent.

4- hydrochlorides (DAB).

Commented [QA25]: At what phase (beginning ofstationary phase or in the stationary phase, etc.) was IPTG

3．2.3 Sequence aAnalysis of the sequence
The positive plaques for reactive against A. lumbricoides antibody antibodies wereadded to the membrane. This is an important aspect of
induction protocol that needs to be clarified.

picked up and transformed into anthe E.coli host. These clones were constructed as aplasmid, pExcell. The sequences of positive clones were analyzed using the BigDye

Commented [QA26]: Is the plasmid sequence for pExcell

Terminator v3.1 cycle sequencing kit (Becton Dickinson Biosciences). The plasmid

published? If so please cite it in this section.

sequences were then compared with the sequences in the GenBank database (National
Institute for Biotechnology Information) to determine the identity of genes.
4.2.4 Identifying the site of mucin12mucin12 localization in A. lumbricoides
The A 14 amino acid synthetic peptide of 14 amino acid (HREQYDVPQEWRKE)

Formatted: Not Highlightfrom amino acid 396 to 409 of mucin12mucin12 were was synthesized and administered

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it to rabbits to prepare anti-mucin12mucin12 polyclonal antibody antibodies by Sigma-

the care and use of animals and of experimental

Ggenosys (Hokkaido, Japan). On day 0, The first administration to the rabbit on day 0

procedures.

was given a 200 µg dose of the peptide, then boosted by followed by an additional 100 µg
in 5 subsequent administrations on days 7, 14, 21, 27, and 42 with incomplete Freud ‘s ’s
adjuvant. On day 49, exsanguination was conducted. Furthermore, tThe serum waspurified by ammonium sulfate precipitation.
To identify the localization of mucin12 in A. lumbricoides, we conducted immunostaining with sections of A. lumbricoides. Three frozen sections were used: one from theCommented [SE28]: The “Ethical approval” aspect of
this paper is blank. Approval for the use of rabbits forgeneration of polyclonal serum and human tissue forstaining of mucin isoforms is not described in thematerials and methods. No major journal will accept thesestudies without statement of ethical approval.

head, the quarter point offrom the head, and the quarter point from of the tail of A.

lumbricoides. The sections of A. lumbricoides were cut into sliced with μ ofthicknessslices and stained with HE. In order to identify the localization of mucin12 in

A. lumbricoides, we conducted immuno-staining with sections of A. lumbricoides. After
blocking, the sections were stained with the polyclonal anti-mucin12mucin12
antibodiesy (×x150 dilution) and mucin12 antibody antibodies after the absorption
treatment with the synthetic peptide described above earlier in this section to identify

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Commented [SE29]: “The quarter point” does not makeit clear where the sections were taken from. Please clarify.
Commented [QA30]: How thick were these sections cut
for immunostaining? Was the polyclonal antibody to
Muc12 validated in any other way independent of peptide
absorption?

the specificity of the primary antibody. After washing with PBS with Tween 20, FITC
anti-rabbit IgG (Sigma-Aldrich, ×x150 dilution) were was used for as the secondary
antibody. These sections were observed by using a laser confocal microscope LSM510(Carl Zeiss).
To confirm the mucin12 existing in A. lumbricoides crude antigen, SDS-PAGE
was performed using a crude extract of A. lumbricoides. For western -blotting, crude

Commented [SC ED31]: Please specify the quantity of

extract was transferred onto nitrocellulose membrane by a semidry transfer system

crude extract used.

(ATTO, Japan) and incubated with anti-mucin12 polyclonal antibodiesy (×x1,000
dilution) for primary antigen after blocking. Furthermore, theThe membrane wasincubated with HRP aAnti-rabbit IgG for the secondary antibodiesy and the reacted

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bands were visualized by using the ECL method (GE healthcare) using and X-ray films.

company from where you have purchased this anti-

In order tTo confirm that the results of immunoe-staining were not affected bymucin12mucin12 that were derived fromof human origin, wWestern -blotting was

immunoglobulin. Also, provide the dilution of theantibody used in this experiment.

performed with protein extracts of A. lumbricoides crude antigen and mouse Embryonic
Stem (ES) cell lines using RaAnti-GAPDH polyclonal antibodiesy that could cross-react

Commented [SC ED33]: Please mention the source towith some mammalian GAPDH (human, mouse, and rat, etc.) (Cat: ab9485-25, Abcam,

which this polyclonal antibody belongs.

×x1,000) for the primary antibodiesy and HRP anti-rabbit IgG (Sigma-Aldrich, as
described above) for the secondary antibodiesy.
3. Results
1.3.1 Screening with against a human cDNA libraryCommented [SE34]: Please describe how the screening

As the results of homology search, tAhe fragments of 389 bp wasof “clones” or “plaques” was done at the beginning of theidentifiedmatched with as frombase 756 bases to 1144 base of human transmembrane

results section to help clarify the data in this section withmucin12mucin12 (AF147790) with and its consistent was 99.5% consistency (Fig. 1).

the reader.

[Other text deleted]
2.3.2 Identifying the site of mucin12 localization in A. lumbricoides

In order to identify the localization of mucin12mucin12 inon A. lumbricoides,
immunohistochemistry was performed with the anti-mucin12mucin12 polyclonal
antibodiesy.

As the results, the FITC- labeled signals were detected in the mucous

secretory organs, epidermis, and intestinal canal (Fig. 2b and 2c). In order tTo confirm

Formatted: Not Highlightthe specificity of the antibody, the mucin12mucin12 antibodyies was were pre-absorbedwith a synthetic peptide that was used for the preparation of polyclonal antibodiesy anddone the staining as samestained as described abovein section 2. As showned in Fig. 2d

Commented [SE35]: Should this perhaps be in sectionand 2e, the signals were clearly disappeared. These results were suggested that the2? Please check.

protein which that reacts with anti-human mucin12mucin12 is localized in the mucous
secretory organs, epidermis, and intestinal canal in of A. lumbricoides.

Commented [QA36]: Although you have indicated thelocations of the protein reactive mechanism in the[Other text deleted]parasite, it would be ideal to emphasize on which of theselocations showed the most promising outcome.

4. Discussion

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It was discussedhas been suggested that one of an the parasite’s immunologicalescape mechanism utilized bys A. lumbricoides is the expression of the matteran antigenvery similar to to itsa host antigen [6]. However, the parasite’s immunological escapemechanisms have have not yet been sufficiently clearly unraveled yetelucidated, so thishypothesis has not been definitively confirmed.

Therefore, the researchers authors of

the present report decided to reveal examine the this potential immunological escapemechanism using a molecular biological biology tool.

After

conductingascreening against a human colon cDNA library of human colon using anti- A.

lumbricoides polyclonal antibody antibodies and checking for a common antigen, 3 threepositive clones were obtained.

One of the rResults of the analysis of each sequence

showed that one clone possessed high homologous homology with transmembrane mucin
12mucin12.

Commented [QA37]: Although your meaning is

Mucins are mucous glycosylated proteins that are important components ofsufficiently evident through this paragraph, I havemucous that covering inner cavities such as the trachea, the digestive tract including theinserted further edits in this section to depict the core

stomach and intestines, and the gonads [11].

intended research approach of this study in comparison toexisting literature. Also, it not very well correlates to the[Other text deleted]finding of 3 positive clones after screening of the cDNA

At the time of writingCurrently, 18 different mucin genes proteins have beenreported to exist in humans.

There are secreted mucins, which are secreted from

epithelial cells, and membrane-associated mucins, which have transmembrane sections
and exist under cellular membrane-bound conditions.
We examined transmembrane mucin12 In order to observe thein the context

library.
Commented [SE38]: This sentence has been deleted to
avoid redundancy.
Formatted: Not Highlightassociation of transmembrane mucin12 inof the rest of the mucin family., Ggenomic

Commented [QA39]: The intended importance of the

mucin12 DNA can be found on chromosome 7, along with that of of mucin12 including interm ‘transmembrane’ should be explained further

mucin 3 and mucin 17 are existed on same chromosome 7 [20], and mucin12mucin12

because scientists have explored the membraneprotein is comprised of comprises a total of 588 amino acid sequences.

properties in context of the parasite’s ability to defy the

Mucin 12Mucin12 was localized in the mucous secretory organs and thehost’s immune mechanism. This aspect should be clearly

epidermis of hypodermis of A. lumbricoides by immunostaining with mMucin12ucin12

depicted by you through these lines.

polyclonal antibody (Fig.

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The mucous secretory organs are connected to theepidermis of hypodermis and the lateral cord, and are involved in excretion and form theCommented [SE40]: Please provide the figure number.

cluster-like .[21]. There is an excretory canal in the lateral cord, and it is connected toFormatted: Not Highlightthe surface of the worm body.

Commented [QA41]: This explanation about lateral cord

Then, wWestern blotting was conducted to examine the existence of common
antigenicity of mucin12mucin12 in A. lumbricoides crude antigen.

A band was detected

does not add significance to the explanation you haveprovided about mucous secretory organs in relation toaround 37 kDa. Additionally, a specific band was detected in the protein extracted frommucin12. We have thus deleted it to make the paragraphcultured mouse cell but not in human A. lumbricoides crude antigen.

more concise and focussed.

Researchers The authors would like to further examine the protein, which iscross-reactive with mucin12mucin12 in the A. lumbricoides crude antigen, and thematter substance similar to actin and beta-casein-like-protein detected asnoted to havecommon antigenicity, both detected in this study. We would also likeAnd to researchCommented [SE42]: Is it known what the sequence ofthe 37KDa protein from the helminth is and how it ismodified (glycosylated or a receptor) during infection?

which investigate whether any A. lumbricoides hosts areis immune to avoidance byCommented [QA43]: This is a non-specific expression

mucin 12mucin12,and should be elaborated with proper context.

of the mechanism and the other mechanisms of immune avoidance

utilized by A. lumbricoides.

Formatted: Not Highlight
Commented [SE44]: Please check if the edit retains your5. Conclusion
In this study, analysis of common antigenicity between A. lumbricoides andintended meaning.
Commented [QA45]: There are papers that haveintestinal mucosa obtained three DNA clones. After analyzingAnalysis of each clone

reported about expression of mucin proteins being one ofsequence indicated , it was clarified that one of them has high homology withthe pathogenic mode of overcoming the antigenic defencetransmembrane mucin12mucin12.

mechanism of the host. Hence, this concluding remark

Localization of mucin12mucin12 was confirmed in the mucousmucosal

should be more focussed on how your findings canepithelial present around the intestinal crypts and villi of the human small intestine.

contribute to the next level of analysis by other

These data suggest that expression of mucin proteins by helminths may be one

researchers in this field.

mechanism by through which the helminth parasite evades immunological detection

You might probably refer to the continuous evolution ofwithin the mammalian host.

parasitic immune regulatory pathways that can be
explored towards obtaining a novel therapeutic outcome.

6. Acknowledgments
Ethical approval

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those individuals who provided help during the research(e.g., providing language help, writing assistance or proof...

[Other text deleted]
Figure lLegends:

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Figure 1. Sequence analysis of a newly found identified clone after screening of of the

included in the single file are placed next to the relevant

A. lumbricoides cDNA library. TM12: human transmembrane mucin 12mucin12.

text in the manuscript, rather than at the bottom or the
top of the file. The corresponding caption should be

Figure 2.

Identification of the lLocalization for of mMucin12ucin12 in human the A.

Lumbricoides lumbricoides adult worm. The sSections of A. Lumbricoides lumbricoides
were stained with HE (a) and anti-mMucin12ucin12 polyclonal antibodiesy . The sectionsof A. lumbricoides were stained with anti-Mucin12 polyclonal antibody that was pretreatedpretreated with synthetic mMucin12ucin12 peptide (d and e). The scale bar
indicates represents 200 µm (b and d), and 100 µm (a, c and e), respectively.

placed directly below the figure or table.