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\begin{document}

\title{Function and mechanism study of the lncRNA uc.375 for arrest of alveolar
development in bronchopulmonary dysplasia}



\author[1]{Tianping Bao}%
\author[2]{Haiyan Zhu}%
\author[2]{Yafei Zheng}%
\author[2]{jingjing hu}%
\author[2]{Huifang Wang }%
\author[2]{huaiping         Cheng }%
\author[2]{yuan zhang}%
\author[3]{tian zhaofang}%
\affil[1]{Nanjing Medical University}%
\affil[2]{The Affiliated Huaian No.1 People's Hospital of Nanjing Medical University}%
\affil[3]{Huaian first hospital,Nanjing Medical University  }%


\vspace{-1em}



  
  \date{May 20, 2024}


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\selectlanguage{english}
\begin{abstract}
we identified a molecular effector mechanism of the lncRNA uc.375 in
lung tissue of BPD using lncRNA microarray, bioinformatics, and
functional studies. We found that uc.375 is mainly distributed in AEC
II, and low expression in BPD mice AEC II. Silencing uc.375 promoted the
apoptosis of MLE 12 cells, reduced their proliferation and
differentiation, and promoted the apoptosis-related factor caspase 3,
inhibited the expression of MLE 12 specific secretion SP-C, bcl2 and
UCP2. On the contrary, after overexpressing uc.375, the opposite results
were obtained. Silencing uc.375 while silencing FoxA1 inhibited MLE 12
apoptosis, promoted their proliferation, inhibited apoptosis-related
factor caspase3, and promoted the expression of MLE 12-specific
secretions SP-C , bcl2 and UCP2. On the contrary, overexpression of
uc.375 and FoxA1 at the same time, the result is opposite. uc.375
negatively regulates FoxA1 expression, affects alveolar development, and
plays an important role in the occurrence and development of BPD. In
summary, this study clarified the exact mechanism of uc.375 in the
occurrence and development of BPD. we identified that it may provide a
new molecular target for the prevention and treatment of BPD.%
\end{abstract}%



\sloppy


\textbf{1. INTRODUCTION}

Bronchopulmonary dysplasia (BPD) is one of the common chronic lung
diseases in preterm infants, and its incidence is as high as 52\% in
extremely low birth weight infants \textsuperscript{1, 2}. With the
increasing maturity of critical care and management techniques for
premature infants, the survival rate of very low and extremely low birth
weight infants has increased significantly, but with it, the incidence
of BPD has also shown a continuous upward trend \textsuperscript{3}.
Children with this disease not only suffer from repeated respiratory
infections and varying degrees of pulmonary dysfunction, but also
increase the risk of asthma and chronic obstructive pulmonary disease
(COPD) in adulthood \textsuperscript{4}. Therefore, BPD has become an
important medical problem that endangers the health of newborns, affects
the quality of our nationals, and causes a heavy family and
socioeconomic burden.

LncRNAs are a type of non-coding transcript that is greater than 200
nucleotides in length and does not have the ability to encode proteins.
In recent years, with the rapid development of genetic information
technology and the wide application of next-generation sequencing and
gene chip technology, scholars' understanding of the pathogenesis of
many human diseases has gradually expanded to the level of lncRNA and
has become a research hotspot in many fields of life medicine. It has
also become a new direction for us to explore the pathogenesis of BPD.
LncRNAs have abundant biological functions in cell development and
metabolism, such as genetic imprinting \textsuperscript{5}, chromatin
modification \textsuperscript{6}, cell cycle
regulation\textsuperscript{7}, transcription regulation
\textsuperscript{8}, and participation in mRNA degradation
\textsuperscript{9} Etc. These are closely related to the occurrence,
development and prevention of many human diseases. Studies on the role
and mechanism of lncRNA in lung development and lung diseases have
become a hot spot of increasing attention by scholars. Herriges MJ et
al. \textsuperscript{10}identified 363 lncRNAs in embryonic mouse lungs,
which interact with transcription factors spatially to regulate lung
development. The lack of certain lncRNAs in the lungs can lead to
alveolar capillary dysplasia and pulmonary vein dislocation (ACD/MPV)
\textsuperscript{11}. Studies have also confirmed that lncRNA plays the
role of ''inducers and terminators'' of vascular development
\textsuperscript{12}, affects the biological functions of endothelial
cells, and can be used as a target for vascular diseases
\textsuperscript{13}. Although there have been preliminary studies of
lncRNA in the field of lung development, further exploration of the role
and mechanism of lncRNA in the formation of BPD will inevitably promote
neonatal scholars' in-depth understanding of the pathogenesis of BPD.

In this study, we identified a molecular effector mechanism of the
lncRNA uc.375 in lung tissue of BPD using lncRNA microarray,
bioinformatics, and functional studies. We found that uc.375 is mainly
distributed in AEC II, and low expression in BPD mice AEC II. Silencing
uc.375 promoted the apoptosis of MLE 12 cells, reduced their
proliferation and differentiation, and promoted the apoptosis-related
factor caspase 3, inhibited the expression of MLE 12 specific secretion
SP-C, bcl2 and UCP2. On the contrary, after overexpressing uc.375, the
opposite results were obtained. Silencing uc.375 while silencing FoxA1
inhibited MLE 12 apoptosis, promoted their proliferation, inhibited
apoptosis-related factor caspase3, and promoted the expression of MLE
12-specific secretions SP-C , bcl2 and UCP2. On the contrary,
overexpression of uc.375 and FoxA1 at the same time, the result is
opposite. uc.375 negatively regulates FoxA1 expression, affects alveolar
development, and plays an important role in the occurrence and
development of BPD. In summary, this study clarified the exact mechanism
of uc.375 in the occurrence and development of BPD. we identified that it
may provide a new molecular target for the prevention and treatment of
BPD.



\textbf{2. METHODS}

\textbf{2.1 qRT-PCR}

Total RNA was isolated from tissues and cells using TRIzol Reagent
(Applygen, Beijing, China) in accordance with the manufacturer's
instructions. qRT-PCR was carried by using Reverse Transcription Kit
(Haigene, Harbin, China). After reverse transcription, quantitative
real-time PCR analysis was performed as previously
described\textsuperscript{14}.

\textbf{2.2 Cell culture and transfection}

MLE 12 cell line was obtained from Bei Na Chuang Lian Biotechnology
Research Institute (Beijing, China) and followed their instructions to
culture at 37\selectlanguage{ngerman}°C. Cell transfection was conducted following the
instruction of Lipofectamine 3000 (Invitrogen, CA, USA). Stably uc.375
or FoxA1 silencing cell lines were screened out as previously
reported\textsuperscript{15}. The cells were transfected with 10 pmol/mL
of uc.375 or FoxA1 short-hairpin RNA, or negative control
(Sigma-Aldrich) and selected for 4 weeks with neomycin (1000 \selectlanguage{greek}μ\selectlanguage{english}g/ml). To
up-regulate uc.375 or FoxA1, the MLE 12 cells were transfected with
uc.375 or FoxA1 overexpression plasmid according to the manufacturer's
protocols.

\textbf{2.3 CCK-8 assay}

Cell viability was detected by CCK8 kit (MedChemExpress, Shanghai,
China). Cells (3000 / well) were cultured in 96-well plates for 48
hours. After incubated with 10 \selectlanguage{greek}μ\selectlanguage{english}L CCK8 for 3-4 hours, the absorbance was
measured at 450 nm using GloMax\selectlanguage{ngerman}® System (Promega, WI, USA)
after\textsuperscript{16}.

\textbf{2.4 Hoechst PI staining}

The single cell suspension was prepared routinely, the final
concentration of Hoechst 33258 was 1 MG/ML, the final concentration of
Hoechst 33258 was dissolved in water at 37 °C for 7 minutes, the dye
solution was cooled on ice, the dye solution was centrifuged and PBS was
overhung, the final concentration of PI solution was 5 MG/ML, the ice
bath was added, the dye solution was centrifuged and PBS was washed
once, the Blue fluorescence at 400 \textasciitilde{} 500 nm and the red
fluorescence at 630 nm or above were recorded by direct external laser
Flow cytometry analysis.

\textbf{2.5 Western blot}

Total proteins were isolated from tissues using a total protein
extraction kit (Keygen, Nanjing, China). A total of 40 \selectlanguage{greek}µ\selectlanguage{english}g protein was
separated using SDS-PAGE and transferred onto polyvinylidene difluoride
(PVDF) membranes and then blocked with 5\% fat-free milk at room
temperature for 2 h. The immune-blot was incubated with primary antibody
(1:1000 dilution; Cell signaling) and GAPDH (1:1000 dilution; Cell
signaling) was used as a control. The signals were detected using a
Super ECL Plus Kit (Keygen) and determined by quantitative analysis
using UVP software (UVP, LLC, Upland, CA, United States).

\textbf{2.6 Flow cytometry assay}

The apoptosis rate of MLE12 cells was detected by Annexin V-FITC/PI
Apoptosis Detection Kit (Yeasen, Shanghai, China) via flow cytometry.
After cultured for 48 hours, cells were collected, following by staining
with 10 \selectlanguage{greek}μ\selectlanguage{english}L of Annexin V-FITC and PI. Then cells were analyzed by flow
cytometer (BD, New Jersey, USA) \textsuperscript{17}.

\textbf{2.7 Statistical analysis}

Data are shown as mean \selectlanguage{ngerman}± SD. SPSS 21.0 (IBM Corp., NY, USA) was applied
for statistical analysis of all data. T-test was used for comparison
between two groups, and one-way ANOVA and Tukey's post-tests were used
for multiple groups. The level of significance was set at P \textless{}
0.05.



\textbf{3. RESULTS}

\textbf{3.1 Analysis of uc.375 expression characteristics}

We placed newborn mice in a 95\% high-oxygen environment for 7 days, and
the pathological structure of the mouse lung successfully simulated the
characteristics of human BPD. We selected lung tissue samples of the BPD
group and the air control group as the test objects, and used lncRNA
microarray technology (Mouse lncRNA Microarray V3.0, Arraystar, probes
covering 35923 long non-coding RNAs and 24881 coding genes) to screen
BPD related lncRNAs. The results suggest that the expression of lncRNA
in lung tissue of BPD induced by hyperoxia is quite different from that
in normal lung tissue (Figure 1A. Scattered analysis diagram; Figure 1B.
Cluster analysis), suggesting that these differences in lncRNA may be
the molecular basis for the development of BPD; When analyzing the
differentially expressed lncRNAs, it was found that there were 140
upregulated lncRNAs with 5 times or more, and 71 downregulated lncRNAs
with 5 times or more.

We also conducted a bioinformatics analysis on uc.375 and identified
that uc.375 has a full length of 300bp and is located on human
chromosome 14q13 (Figure 1C). It is well-conserved among mammals (Figure
1D and E). There are many transcription factor binding sites in the
chromosome region where the uc.375 sequence is located (Figure 1F),
suggesting that it may have a potentially powerful transcriptional
regulatory effect. We further investigated the expression of uc.375 at
different time points in the development of BPD. We exposed newborn mice
to 95\% oxygen, real time PCR was used to detect the expression of
uc.375 in lung tissue at different time points (D0, D1, D3, D5, D7 day)
. The results showed that uc.375 decreased gradually with time (Figure
1G). We detected the localization of uc.375 in MLE 12 cells by RNA ISH,
and we found that uc.375 existed in the nucleus (Figure 1H).

\textbf{3.2 The effect of uc.375 silencing on MLE 12 cells function and
FoxA1 expression}

To assess the effect of uc.375 silencing on MLE 12 cells function, MLE
12 cells were stably transfected with uc.375 silent lentiviral vector to
knock down uc.375. CCK-8 assay was conducted and cell viability was
diminished by sh-uc.375 (Figure 2A). In addition, the effect of
sh-uc.375 on cell apoptosis was assessed by Hoechst/PI staining and flow
cytometry. The cell apoptosis was remarkably promoted by sh-uc.375
(Figure 2B and 2D). Moreover, the protein levels of caspase3, FoxA1,
SP-C and UCP2 were investigated by western blot in MLE12 cells. The
results showed that SP-C and UCP2 were suppressed, whereas caspase3 and
FoxA1 were markedly stimulated by sh-uc.375 (Figure 2C). In addition,
the qRT-PCR results confirmed that uc.375 level was significantly
repressed by sh-uc.375, the SP-C and UCP2 mRNA expression were
suppressed, whereas FoxA1 was markedly stimulated by sh-uc.375(Figure
2E-H). Taken together, uc.375 silencing inhibited cell proliferation and
promoted apoptosis.

\textbf{3.3 The effect of uc.375 overexpression on the function of MLE
12 cells}

To further assess the effect of uc.375 overexpression on MLE 12 cells
function, MLE 12 cells were stably transfected with uc.375
overexpression lentiviral vector to overexpress uc.375. CCK-8 assay was
conducted and cell viability was increased by uc.375 overexpression
(Figure 3A). In addition, the effect of uc.375 overexpression on cell
apoptosis was assessed by Hoechst/PI staining and flow cytometry. The
cell apoptosis was remarkably reduced by uc.375 overexpression (Figure
3B and 3D). Moreover, the protein levels of caspase3, FoxA1, SP-C and
UCP2 were investigated by western blot in MLE12 cells. The results
showed that SP-C and UCP2 were increased, whereas caspase3 and FoxA1
were markedly inhibited by uc.375 overexpression (Figure 3C). In
addition, the qRT-PCR results confirmed that uc.375 level was
significantly elevated by uc.375 overexpression, the SP-C and UCP2 mRNA
expression were stimulated, whereas FoxA1 was markedly suppressed by
uc.375 overexpression (Figure 3E-H). Taken together, uc.375
overexpression promoted cell proliferation and repressed apoptosis.

\textbf{3.4 Effects of uc.375 silencing and FoxA1 silencing on the
function of MLE 12 cells}

To further evaluate the effect of uc.375 silencing and FoxA1silencing on
MLE 12 cells function, MLE 12 cells were stably co-transfected with
uc.375 silencing/FoxA1 silencing to knock down uc.375 and FoxA1. CCK-8
assay was conducted and cell viability was increased by FoxA1 silencing
(Figure 4A). In addition, the cell apoptosis was assessed by Hoechst/PI
staining and flow cytometry. The cell apoptosis was notably reduced by
FoxA1 silencing (Figure 4B and 4D). Moreover, the protein levels of
caspase3, FoxA1, SP-C and UCP2 were detected by western blot in MLE12
cells. The results showed that SP-C and UCP2 were increased, whereas
caspase3 and FoxA1 were markedly inhibited by FoxA1 silencing (Figure
4C). In addition, the qRT-PCR results confirmed that uc.375 level was
significantly reduced by uc.375 silencing, the SP-C and UCP2 mRNA
expression were stimulated, whereas FoxA1 was markedly suppressed by
FoxA1 silencing (Figure 4E-H). Taken together, FoxA1 silencing
stimulated cell proliferation and suppressed apoptosis.

\textbf{3.5 Effects of uc.375 overexpression and FoxA1 overexpression on
the function of MLE 12 cells}

To further evaluate the effect of uc.375 overexpression and FoxA1
overexpression on MLE 12 cells function, MLE 12 cells were stably
co-transfected with uc.375 overexpression /FoxA1 overexpression to
overexpress uc.375 and FoxA1. CCK-8 assay was conducted and cell
viability was decreased by FoxA1 overexpression (Figure 5A). In
addition, the cell apoptosis was assessed by Hoechst/PI staining and
flow cytometry. The cell apoptosis was notably increased by FoxA1
overexpression (Figure 5B and 5D). Moreover, the protein levels of
caspase3, FoxA1, SP-C and UCP2 were detected by western blot in MLE12
cells. The results showed that SP-C and UCP2 were reduced, whereas
caspase3 and FoxA1 were markedly increased by FoxA1 overexpression
(Figure 5C). In addition, the qRT-PCR results confirmed that uc.375
level was significantly elevated by uc.375 overexpression, the SP-C and
UCP2 mRNA expression were inhibited, whereas FoxA1 was markedly promoted
by FoxA1 overexpression (Figure 5E-H). Taken together, FoxA1
overexpression suppressed cell proliferation and stimulated apoptosis.

\textbf{4. DISCUSSION}

At present, the pathogenesis of BPD has not yet been fully elucidated.
Most scholars believe that BPD is a multifactorial disease-causing
syndrome. Type II epithelial cells (alveolar epithelial II cells, AEC
II) apoptosis dominated immature lung injury, its pathological feature
is alveolar developmental block with increased alveolar epithelial cell
apoptosis \textsuperscript{18, 19}. AEC II is a stem cell of alveolar
epithelial cells, which has the ability of immortal proliferation and
differentiation \textsuperscript{20, 21}. During lung development, AEC
II continuously differentiates into alveolar epithelial cell I (AEC I)
to promote alveolar formation and complete alveolar remodeling after
epithelial injury \textsuperscript{22}. AEC II is an important effector
cell in the pathological process of lung injury. Its function and status
determine the pathological outcome of lung injury; AEC II's survival and
apoptosis affect the outcome of lung injury repair.

Lung development is an intricate process. From a genetic perspective, it
is the result of the expression, regulation and interaction of key lung
development genes at different times and in different spaces. A slight
abnormality in any link may disrupt normal lung development and cause
varying degrees lung injury \textsuperscript{23}. With the rapid
development of molecular genetics research, a number of genes closely
related to BPD have been discovered, such as VEGF-\selectlanguage{greek}α, \selectlanguage{english}TGF-\selectlanguage{greek}β, \selectlanguage{english}IGF-1,
fibronectin 1, p21, FoxA1, etc. The expression level of these genes in
lung tissues To a certain extent, the changes in BPD can reflect the
development trend of BPD \textsuperscript{23}. Studies have reported
that non-coding RNA is also involved in the pathogenesis of BPD. For
example, miRNA-29 is highly expressed in the lung tissue of newborn mice
with BPD, and it participates in the occurrence and development of BPD
by down-regulating the expression of Ntrk2 and disrupting various
biological processes of lung development \textsuperscript{24}; Recent
studies have also reported that lncRNA MALAT1 can protect BPD by
inhibiting cell apoptosis \textsuperscript{25}. Although scholars at
home and abroad have done a lot of meaningful work on the pathogenesis
of BPD, providing molecular basis and clues for understanding the
formation process of BPD, the pathogenesis of BPD is still inaccurate,
and the existing pathogenesis cannot provide it. Effective clinical
interventions, so in-depth exploration of new mechanisms of BPD
pathogenesis is urgent.

uc.375 is a lncRNA with unknown function that is highly conserved among
different species, differentially lowly expressed in BPD lung tissue,
localized in alveolar type II epithelial cells, and obtained through
microarray technology screening. Regarding the biological information,
function and role of uc.375 in BPD, there are currently no relevant
reports at home and abroad, and there is no report on the function and
mechanism of uc.375 involved in the process of BPD alveolar development.
Our research found that silencing uc.375 in the mouse alveolar type II
epithelial cell line (MLE 12) can significantly promote its apoptosis
and inhibit its proliferation; uc.375 negatively regulates the
expression of FoxA1, affects the development of alveoli, and occurs in
BPD. Play an important role in development.

The FoxA family is a class of proteins with important functions, which
are involved in the regulation of cell proliferation, differentiation,
embryonic development and other important life activities. Some members
of the FoxA family play an important role in the regulation of
apoptosis-related genes. For example, FoxA2 can significantly inhibit
the expression of SP-A in A549, and SP-A is an apoptosis inhibitor of
alveolar epithelial cells \textsuperscript{26}; 2-Acetylaminofluorene
promotes bile duct cell apoptosis through FoxA3 \textsuperscript{27}.
FoxA1's nuclear localization sequence, DNA binding domain, and NH2
terminal transcription activation are highly homologous to FoxA2 and 3.
FoxA1 is closely related to the embryonic development of
endoderm-derived tissues and organs. Lee CS et al.
\textsuperscript{28}revealed that the initiation of liver development
during embryonic development is FoxA1-dependent, and the development of
fetal lungs is also regulated by FoxA1, and FoxA1 is only found in the
lungs. It is expressed in airway epithelial cells and alveolar type II
epithelial cells in tissues \textsuperscript{29}. Studies have also
found that the loss of FoxA1 in mouse lung tissue AEC II and bronchial
Clara cells will delay cell development and maturation. In addition, the
expression of SP-B and SP-C in mice lacking FoxA1 is significantly lower
than that in wild-type mice, eventually leading to increased
susceptibility to respiratory diseases after birth \textsuperscript{30,
31}. It is more reported in the literature that FoxA1 inhibits its
expression by combining with the anti-apoptotic genes bcl2 and FoxA1
binding elements in the promoter region of UCP2, thereby promoting AEC
II apoptosis and playing a role in BPD \textsuperscript{32}. In this
study, we identified that FoxA1 is the regulatory target of uc.375.

\textbf{5. CONCLUSION}

In summary, our findings elucidated that uc.375 negatively regulates
FoxA1 expression, affects the development of alveoli, and plays an
important role in the occurrence and development of BPD. It will provide
Our study provides a novel idea and potential target for further study
on prevention and treatment of BPD.

\textbf{ETHICS STATEMENT}

All animal experiments were conducted with the approval of the
Institutional Animal Care \& Use Committee of the Affiliated Huaian No.1
People's Hospital of Nanjing Medical University.

\textbf{AUTHOR CONTRIBUTIONS}

TB and ZT conceived the project and designed the experiments. TB
performed the study, analyzed the data, and wrote the paper. HZ, YZ, JH,
HW, HC and YZ performed the study. TB and HZ analyzed the data. ZT
analyzed the data and critically revised the manuscript.

\textbf{Consent for publication}

All authors agree with the content of the manuscript.

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\textbf{Figure Legends}

\textbf{Figure 1 Analysis of uc.375 expression characteristics.} A.
Scatter analysis chart of lncRNAs expression in hyperoxia-induced BPD
lung tissue and normal lung tissue. B. Cluster analysis. C-F. uc.375
Bioinformatics characteristics. G. The relative mRNA expression level of
uc.375 was detected by realtime PCR in lung tissues at different time
points (D0, D1, D3, D5, D7 days) during BPD modeling. H. The
localization of uc.375 in MLE 12 cells.

\textbf{Figure 2 The effect of uc.375 silence on MLE 12 cells function
and FoxA1 expression.} A. The proliferation activity of MLE 12 cells was
detected by CCK-8 assay. B. The cell apoptosis was determined by
Hoechst/PI staining in MLE12 cells. C. The protein levels of
Cleaved-caspase3, FoxA1, SP-C and UCP2 were investigated by western blot
in MLE12 cells. D. The cell apoptosis was determined using low cytometry
assay in MLE12 cells. E-H. The relative mRNA expression levels of
uc.375, FoxA1, SP-C and UCP2 were detected by realtime PCR in MLE12
cells. *\emph{P} \textless{} 0.05, **\emph{P} \textless{} 0.01\emph{vs}
. sh-Con.

\textbf{Figure 3} \textbf{The effect of uc.375 overexpression on the
function of MLE 12 cells.} A. The proliferation activity of MLE 12 cells
was detected by CCK-8 assay. B. The cell apoptosis was determined by
Hoechst/PI staining in MLE12 cells. C. The protein levels of caspase3,
FoxA1, SP-C and UCP2 were investigated by western blot in MLE12 cells.
D. The cell apoptosis was determined using low cytometry assay in MLE12
cells. E-H. The relative mRNA expression levels of uc.375, FoxA1, SP-C
and UCP2 were detected by realtime PCR in MLE12 cells.
*\emph{P}\textless{} 0.05, **\emph{P} \textless{} 0.01 \emph{vs} .
sh-Con.

\textbf{Figure 4 Effects of uc.375 silencing and FoxA1 silencing on the
function of MLE 12 cells.} A. The proliferation activity of MLE 12 cells
was detected by CCK-8 assay. B. The cell apoptosis was determined by
Hoechst/PI staining in MLE12 cells. C. The protein levels of caspase3,
FoxA1, SP-C and UCP2 were investigated by western blot in MLE12 cells.
D. The cell apoptosis was determined using low cytometry assay in MLE12
cells. E-H. The relative mRNA expression levels of uc.375, FoxA1, SP-C
and UCP2 were detected by realtime PCR in MLE12 cells. *P \textless{}
0.05, **P \textless{} 0.01 vs. sh-Con.

\textbf{Figure 5 Effects of uc.375 overexpression and FoxA1
overexpression on the function of MLE 12 cells.} A. The proliferation
activity of MLE 12 cells was detected by CCK-8 assay. B. The cell
apoptosis was determined by Hoechst/PI staining in MLE12 cells. C. The
protein levels of caspase3, FoxA1, SP-C and UCP2 were investigated by
western blot in MLE12 cells. D. The cell apoptosis was determined using
low cytometry assay in MLE12 cells. E-H. The relative mRNA expression
levels of uc.375, FoxA1, SP-C and UCP2 were detected by realtime PCR in
MLE12 cells. *P \textless{} 0.05, **P \textless{} 0.01 vs. sh-Con.\selectlanguage{english}
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