LncRNA-PANDAR regulates the progression of thyroid carcinoma by targeting miR-637/KLK4.

Thyroid gland carcinoma (TC) originates from follicular or parafollicular thyroid cells and is one of the most common endocrine organ malignancies. To explore the molecular mechanism by which long-chain non-coding RNAs regulate the growth and metastasis of thyroid gland carcinoma, in this study we focused on long non-coding RNAs (lncRNAs) that have been reported to be involved in tumorigenesis. We identified Promoter Region of CDKN 1A antisense DNA damage-activated RNA (PANDAR), which was positively correlated with thyroid gland carcinoma risk. PANDAR could promote thyroid gland carcinoma cell proliferation and metastasis. PANDAR negatively correlated with miR-637, and miR-637 overexpression suppressed thyroid gland carcinoma progression, which could be reversed by PANDAR. MiR-637 could target Kallikrein-related peptidases 4 (KLK4) to inhibit its expression, which was high in thyroid gland carcinoma. KLK4 inhibited cell progression in thyroid gland carcinoma cells. Knockdown of PANDAR expression inhibited cancer progression in nude mice. Overall, PANDAR can suppress miR-637 and induce KLK4 to regulate invasion and migration in thyroid gland carcinoma. Additionally, we identified miR-637 as a target of PANDAR in thyroid gland carcinoma, and PANDAR can be used as a novel therapeutic target for the treatment of thyroid gland carcinoma.

Association of genetic and immuno-characteristics with clinical outcomes in patients with RET-rearranged non-small cell lung cancer: a retrospective multicenter study.

BACKGROUND: Rearranged during transfection (RET) has been proven to be a tumorigenic target in non-small cell lung cancers (NSCLCs). In RET-rearranged NSCLCs, molecular features and their impact on prognosis were not well illustrated, and the activity of mainstay therapeutics has not currently been well compared. METHODS: Patients diagnosed with NSCLCs with RET rearrangements were analyzed for concomitant mutations, tumor mutation burden (TMB), PD-L1 expression, T cell receptor repertoire and clinical outcomes with chemotherapy, immune checkpoint inhibitors (ICIs), and multikinase inhibitors (MKIs). RESULTS: Among 129 patients with RET-rearranged NSCLC who were analyzed, 41.1% (53/129) had co-occurring genetic alterations by next-generation sequencing, and concomitant TP53 mutation appeared most frequently (20/53, 37.7%). Patients with concurrent TP53 mutation (n = 15) had shorter overall survival than those without (n = 30; median, 18.4 months [95% CI, 8.6-39.1] vs 24.8 months [95% CI, 11.7-52.8]; P < 0.05). Patients with lower peripheral blood TCR diversity (n = 5) had superior overall survival compared with those with higher diversity (n = 6; median, 18.4 months [95% CI, 16.9-19.9] vs 4.8 months [95% CI, 4.5-5.3]; P = 0.035). An association with overall survival was not observed for PD-L1 expression nor for tumor mutation burden level. Median progression-free survival was not significantly different across chemotherapy, ICIs, and MKIs (median, 3.5 vs 2.5 vs 3.8 months). For patients treated with ICIs, the disease control rate was 60% (6/10) and the objective response rate was 20% (2/10). CONCLUSIONS: RET-rearranged lung cancers can be heterogeneous in terms of concomitant genetic alterations. Patients with concurrent TP53 mutation or high peripheral blood TCR repertoire diversity have relatively inferior overall survival in this series. Outcomes with traditional systemic therapies in general are suboptimal.

GADD45α sensitizes cervical cancer cells to radiotherapy via increasing cytoplasmic APE1 level.

Radioresistance remains a major clinical challenge in cervical cancer therapy. However, the mechanism for the development of radioresistance in cervical cancer is unclear. Herein, we determined that growth arrest and DNA-damage-inducible protein 45α (GADD45α) is decreased in radioresistant cervical cancer compared to radiosensitive cancer both in vitro and in vivo. In addition, silencing GADD45α prevents cervical cancer cells from undergoing radiation-induced DNA damage, cell cycle arrest, and apoptosis. More importantly, our data show that the overexpression of GADD45α significantly enhances the radiosensitivity of radioresistant cervical cancer cells. These data show that GADD45α decreases the cytoplasmic distribution of APE1, thereby enhancing the radiosensitivity of cervical cancer cells. Furthermore, we show that GADD45α inhibits the production of nitric oxide (NO), a nuclear APE1 export stimulator, by suppressing both endothelial NO synthase (eNOS) and inducible NO synthase (iNOS) in cervical cancer cells. In conclusion, our findings suggest that decreased GADD45α expression significantly contributes to the development of radioresistance and that ectopic expression of GADD45α sensitizes cervical cancer cells to radiotherapy. GADD45α inhibits the NO-regulated cytoplasmic localization of APE1 through inhibiting eNOS and iNOS, thereby enhancing the radiosensitivity of cervical cancer cells.

3D Topography of the Young Adult Anal Sphincter Complex Reconstructed from Undeformed Serial Anatomical Sections.

BACKGROUND: Pelvic-floor anatomy is usually studied by artifact-prone dissection or imaging, which requires prior anatomical knowledge. We used the serial-section approach to settle contentious issues and an interactive 3D-pdf to make the results widely accessible. METHOD: 3D reconstructions of undeformed thin serial anatomical sections of 4 females and 2 males (21-35y) of the Chinese Visible Human database. FINDINGS: Based on tendinous septa and muscle-fiber orientation as segmentation guides, the anal-sphincter complex (ASC) comprised the subcutaneous external anal sphincter (EAS) and the U-shaped puborectal muscle, a part of the levator ani muscle (LAM). The anococcygeal ligament fixed the EAS to the coccygeal bone. The puborectal-muscle loops, which define the levator hiatus, passed around the anorectal junction and inserted anteriorly on the perineal body and pubic bone. The LAM had a common anterior attachment to the pubic bone, but separated posteriorly into puborectal and "pubovisceral" muscles. This pubovisceral muscle was bilayered: its internal layer attached to the conjoint longitudinal muscle of the rectum and the rectococcygeal fascia, while its outer, patchy layer reinforced the inner layer. ASC contraction makes the ano-rectal bend more acute and lifts the pelvic floor. Extensions of the rectal longitudinal smooth muscle to the coccygeal bone (rectococcygeal muscle), perineal body (rectoperineal muscle), and endopelvic fascia (conjoint longitudinal and pubovisceral muscles) formed a "diaphragm" at the inferior boundary of the mesorectum that suspended the anorectal junction. Its contraction should straighten the anorectal bend. CONCLUSION: The serial-section approach settled contentious topographic issues of the pelvic floor. We propose that the ASC is involved in continence and the rectal diaphragm in defecation.

A population-based study elicits a reverse correlation between age and overall survival in elderly patients with rectal carcinoma receiving adjuvant chemotherapy.

Colorectal cancer is the third most common cancer and the fourth most common cause of cancer-related death globally. This population-based study aimed to explore the predictive factors that affected the overall survival of rectal cancer patients receiving adjuvant chemotherapy plus radical surgery using a Cox proportional hazards modeling approach. A total of 619 patients with rectal cancer who underwent surgery were enrolled between October 2006 and May 2013. Clinical characteristics of the patients were compared among the groups and potential prognostic factors were analyzed using the spss program, version 19.0. Patients aged ≥ 70 years have distinctive characteristics such as lager tumour size (≥ 5 cm), damaged micturition and higher incidence of diabetes compared to younger and middle-aged patients. Male gender, tumour size (≥ 5 cm), poor differentiation, later stage, adjuvant chemotherapy, damaged micturition, hypertension or diabetes are associated with a worse prognosis for rectal cancer patients (P < 0.05). However, smoking is a favourable factor to the patients (P = 0.018). Age of ≥ 70 years is an independent prognostic factor for patients with rectal cancer after surgery (P = 0.000) and elderly patients with Stage II and III disease receiving adjuvant chemotherapy show a favourable prognosis. The elderly patients who suffered from diabetes receiving adjuvant chemotherapy have a poor prognosis. Further prospective and large population studies are warranted to confirm the findings of this study.

APE1 promotes antioxidant capacity by regulating Nrf-2 function through a redox-dependent mechanism.

APE1 is a multifunctional protein that has recently been implicated in protecting cells from oxidative stress. In the current study, we confirmed that APE1׳s effect on cellular antioxidant capacity is related to its redox activity through the use of an APE1 functional mutant, and we investigated the mechanism through which this multifunctional protein affects the function of the transcription factor Nrf-2 in regulating oxidative stress-induced genes. Using a pair of mutants for both the redox activity and the acetylation-regulated activity of APE1, in vitro assays showed that the redox activity of APE1 is crucial for its nuclear association with Nrf-2 and subsequent activation of Nrf-2׳s transcription of several downstream genes during oxidative challenge. Important oxidative stress genes are affected by APE1 redox activity, including Hmox1, Gstm1, and Txnrd1. In addition, utilizing human non-small-cell lung cancer sample tissue as well as a nude mouse xenograft model, we determined that APE1 expression levels are inversely correlated to oxidative stress in vivo. These findings indicated that interference with these crucial functions of APE1 shows promise in preventing resistance to certain radiotherapies and that further research is necessary to understand APE1׳s complex roles in regulating both the basal redox status and the oxidative stress state of the cellular environment.

Genistein alleviates radiation-induced pneumonitis by depressing Ape1/Ref-1 expression to down-regulate inflammatory cytokines.

The aim of the study was to investigate the role of genistein in alleviating radiation-induced pneumonitis(RIP) through down-regulating levels of the inflammatory cytokines by inhibiting the expression of apurinic/apyrimidinic endonuclease 1/redox factor-1 (Ape1/Ref-1). Fifty female C57BL/6J mice (8 weeks old) were randomly divided into a control group, a pure irradiation (IR) group and a genistein + IR group. At the four time points after IR, hematoxylin, and Masson's trichrome stainings were used to examine the pathological changes and collagen fiber deposition. Flow cytometry was used to detect reactive oxygen system (ROS) changes, EMSA was used to estimate the nuclear factor kappa B (NF-κB) transcriptional activities and an ELISA assay was used to measure the levels of TGF-ß1, IL-1ß, TNF-α, and IL-6 in the serum and bronchoalveolar lavage fluid (BALF) 2 weeks after IR.The pathological detection results showed acute inflammatory/fibrinoid exudation of the thoracic tissue after IR,which was significantly alleviated with genistein. The IR inducedan APE1 protein expression increase and NF-jB was effectively suppressed by genistein (P < 0.05). The induction of the inflammatory cytokines TGF-ß1, IL-1ß,TNF-α, and IL-6 by IR were in turn inhibited in the serum and BALF of the genistein-pretreated mice (P < 0.05). In addition, the ROS production was significantly boosted in the A549 cells after IR, which could be down-regulated by the pretreatment of genistein. The results demonstrate that genistein alleviates RIP by attenuating the inflammatory response in the initiation of RIP. A possible target of genistein is the Ape1/ref-1, which regulates key inflammatory cytokines by activating the NF-κB.

FokI polymorphism in vitamin D receptor gene and risk of breast cancer among Caucasian women.

Vitamin D plays a central role in cellular proliferation, apoptosis induction, and tumor growth suppression. The vitamin D receptor (VDR) is a crucial mediator for the cellular effects of vitamin D. A series of epidemiological studies have examined the association between the VDR FokI polymorphism and breast cancer risk, but the findings remain inconclusive. Fifteen eligible case-control studies involving 15,681 cancer cases and 20,632 control subjects were identified through searching PubMed, Embase, and Web of Science. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to assess the association. Heterogeneity across studies was examined with the chi-square-based Q test and the I (2) index. Begg's and Egger's test were also performed to determine publication bias. All statistical data were analyzed by STATA software. The combined estimates did not show significant risks correlated with the FokI polymorphism. However, we found an increased risk in the subgroup analysis by source of control (OR = 1.11, 95% CI = 1.01-1.22; heterogeneity test: P = 0.116, I(2) = 0.0% for ff vs FF; OR = 1.10, 95% CI = 1.01-1.21; heterogeneity test: P = 0.832, I(2) = 0.0% for ff vs Ff + FF). This meta-analysis suggests that the presence of FokI polymorphism may contribute to the risk of breast cancer in Caucasians.

Human apurinic/apyrimidinic endonuclease 1 translocalizes to mitochondria after photodynamic therapy and protects cells from apoptosis.

Photodynamic therapy (PDT) is an effective therapeutic regime for lung cancer. Mitochondrial functional failure is considered to be one of the most important factors causing cell death after PDT. However, the detailed mechanisms that are involved are still unclear. We previously reported that apurinic/apyrimidinic endonuclease (APE1) plays a critical role in regulating sensitivity to PDT in the lung cancer A549 cell line. An important mitochondrial regulatory role for APE1 has recently been reported, so therefore we explored the role of APE1 in cell survival after PDT-induced oxidative stress through regulation of mitochondrial function. We first observed that photoirradiation induced the mitochondrial translocation of APE1. The ability of APE1 to regulate mitochondrial membrane potential and reactive oxygen species (ROS) production after photoirradiation was tested in APE1 knockdown A549 cells. APE1-deficient A549 cells were characterized as having a lower mitochondrial membrane potential and higher ROS production, which led to increased apoptosis through the mitochondrial pathway after PDT. Additionally, unexpected activity of APE1 was observed in mitochondria: the control of mitochondrial transcriptional activity by redox regulation of mitochondrial transcription factor A (TFAM). Furthermore, two dominant-negative mutants of APE1 were overexpressed to enhance their individual activities in mitochondria. The results suggest that both these APE1 activities play a role in the regulation of mitochondrial function but through different mechanisms. The present study not only provides possible mechanisms for APE1 in regulating survival after photoirradiation but also uncovers a new activity of APE1 in mitochondria.

Californium-252 brachytherapy combined with external-beam radiotherapy for cervical cancer: long-term treatment results.

PURPOSE: To observe, by retrospective analysis, the curative effects and complications due to californium-252 (252Cf) neutron intracavitary brachytherapy (ICBT) combined with external-beam radiotherapy (EBRT) in the treatment of cervical cancer. METHODS AND MATERIALS: From February 1999 to December 2007, 696 patients with cervical cancer (Stages IB to IIIB) were treated with 252Cf-ICBT in combination of EBRT. Of all, 31 patients were at Stage IB, 104 at IIA, 363 at IIB, 64 at IIIA, and 134 at IIIB. Californium-252 ICBT was delivered at 7-12 Gy per insertion per week, with a total dose of 29-45 Gy to reference point A in three to five insertions. The whole pelvic cavity was treated with 8-MV X-ray external irradiation at 2 Gy per fraction, four times per week. After 16-38 Gy of external irradiation, the center of the whole pelvic field was blocked with a 4-cm-wide lead shield, with a total external irradiation dose of 44-56 Gy. The total treatment course was 5 to 6 weeks. RESULTS: Overall survival rate at 3 and 5 years for all patients was 76.0% and 64.9%, respectively. Disease-free 3- and 5-year survival rates of patients were 71.2% and 58.4%, respectively. Late complications included vaginal contracture and adhesion, radiation proctitis, radiation cystitis, and inflammatory bowel, which accounted for 5.8%, 7.1%, 6.2%, and 4.9%, respectively. Univariate analysis results showed significant correlation of stage, age, histopathologic grade, and lymph node status with overall survival. Cox multiple regression analysis showed that the independent variables were stage, histopathologic grade, tumor size, and lymphatic metastasis in all patients. CONCLUSION: Results of this series suggest that the combined use of 252Cf-ICBT with EBRT is an effective method for treatment of cervical cancer.

[Identification of antigenic epitopes of human apurinic/apyrimidinic endonuclease monoclonal antibodies and their application].

AIM: To identify the antigenic epitopes of human apurinic/apyrimidinic endonuclease monoclonal antibodies (hAPE1 mAb) and to establish a one-step ELISA for quantitative measurement of hAPE1. METHODS: APE1-15 polypeptide array was used to identify the antigenic epitopes of hAPE1 mAb. Molsoft. ICM-Pro software was used to display the three dimensions of antigenic epitopes of hAPE1 mAbs. hAPE1 mAb was labeled with HRP by modified method of sodium periodate oxidization. A one-step ELISA was established by using two hAPE1 mAbs as a capture antibody and a detection antibody, respectively. RESULTS: The antigenic epitopes of 2-G1 mAb were localized within amino acids (aa) 76-90 and 109-123 in APE1 redox domain, belonging to the conformational epitope category. The antigenic epitope of 4-F6 mAb was within aa 109-147 in DNA repair endonuclease domain. In the range of 8.0 microg/L to 200 microg/L, the hAPE1 antigen showed a good linearity in the standard curve. The minimum detection threshold of this assay was 2.0 microg/L. The average of intra-assay precision and inter-assay precision were 8.67% and 12.45%, respectively. The average recovery rate was 105.47%. CONCLUSION: hAPE1 2-G1 mAb and 4-F6 mAb have different antigenic epitopes, and the one-step ELISA is established successfully to detect the serum hAPE1 level easily, quickly and accurately.

MicroRNA-224 is upregulated in HepG2 cells and involved in cellular migration and invasion.

BACKGROUND AND AIM: MicroRNAs are a class of small non-coding RNAs that negatively regulate the expression of their target genes. The aim of the present study was to explore the effects of microRNA on biological behaviors of HepG2 cells and further analyze its characteristics. METHODS: We detected different expression profiles of miRNAs in HepG2 and L02 cell lines by microRNA microarray. Northern blot, quantitative real-time polymerase chain reaction, methylthiazolyl tetrazolium, fluorescence-activated cell sorting, scratch wound, transwell migration and Matrigel invasion assays and western blot were carried out to determine whether or not microRNA-224 (miR-224) can influence the biological behaviors of HepG2 cells. RESULTS: MiR-224 was significantly upregulated in HepG2 cells. Cell proliferation, migration and invasion, but not cell cycles, were altered after changing the expression of miR-224. Taking invasion and migration as a breakthrough, a close relationship between the expression of miR-224 and its proteins such as PAK4 and MMP9, which were involved in the invasion of tumor, was found. CONCLUSIONS: Overexpression of miR-224 was involved in the malignant phenotype of HepG2 cells, and it may be an important factor in regulating the migration and invasion of HepG2 cells.

[The expression of B-cell translocation gene 2 in diethylnitrosamine-induced primary hepatocellular carcinoma rat model.].

OBJECTIVE: To investigate the expression and role of B-cell translocation gene 2(BTG2) in the carcinogenesis of hepatocellular carcinoma (HCC). METHODS: Modified Diethylnitrosamine (DEN)-induced primary hepatocellular carcinoma rat model was established. The expression of BTG2, p53 and cyclinD1 was detected by RT-PCR, western blot and immunohistochemistry. RESULTS: The BTG2 protein was predominantly localized in the nucleus, with faint cytoplasmic staining in normal liver cells; however, it is mainly a cytoplasmic protein in HCC cells. BTG2 was over-expressed during the early stage after DEN treatment, the expression level peaked at 5 weeks and then it gradually decreased to the normal level after 16 weeks. The expression of cyclin D1 and cyclin E was increased gradually after DEN treatment, and peaked at 16 weeks and 5 weeks respectively. A significant increase in p53 was not observed until 5 weeks after DEN treatment, and it gradually decreased after 16 weeks. CONCLUSIONS: Decreased expression of BTG2 may be an important step in carcinogenesis of HCC. BTG2 may positively regulate p53 expression and negatively regulate cyclin D1 expression in the carcinogenesis of HCC.

[The expression of APE1 and its correlation with prognostic significance after 252Cf radiotherapy in cervical cancer].

OBJECTIVE: To investigate the expression feature of the apurinic/apyrimidinic endonuclease (APE1) and its correlation with clinicopathology and prognostic significance after 252Cf radiotherapy in cervical cancer. METHODS: The expression of APE1 was detected by immunohistochemistry technique in 89 cases of cervical cancer (treated by 252Cf), 15 cases cervical intraepithelial neoplasia (CIN) and 10 cases of normal cervical tissue, and its association with clinicopathological data as well as prognosis were analyzed. RESULTS: The expression of APE1 in cervical cancer is higher significantly than that in normal cervical tissue and CIN (P < 0.01). In normal cervical tissue and CIN, the APE1 express was located in the nucleus. In cervical cancer, the APE1 express was located in the nucleus (59), cytoplasm (8) or nucleus and cytoplasm (22), the location of APE1 was related with FIGO stage and pathological grade (P < 0.01), and not related with lymph node metastasis. The level of APE1 express related with FIGO stage, pathological grade and lymph node metastasis (P < 0.05), and not related with age and pathological type. The Kaplan-Meier survival analysis demonstrated that the survival time of the group of APE1 nucleus expression (median survival time is 70.9 months) and the group of APE1 low expression (median survival time is 75.8 months) is longer significantly than that of the group of APE1 cytoplasm expression (median survival time is 57.8 months) and the group of APE1 high expression (median survival time is 56.5 months) (P = 0.025, 0.001). CONCLUSION: The dystopic express of APE1 might play a pivotal role in carcinogenesis and progression of cervical cancer, and the express of APE1 might estimate the prognosis after 252Cf radiotherapy.

[Follow-up study of clinical effects of californium-252 neutron intracavitary radiotherapy and external beam radiotherapy in endometrial cancer].

OBJECTIVE: To observe the three year local control rate, overall survival rate, complications and prognostic factors of endometrial cancer treated with (252)Cf neutron intracavitary brachytherapy (ICBT) and external beam radiotherapy (EBRT). METHODS: Forty endometrial cancer patients staged Ib - IVa by the standard of Federation of International Gynecologic Organization (FIGO), who had not received any treatment were enrolled in this study. Treatment schedules were: (252)Cf ICBT, 10 - 13 Gy(i)/fraction per week, the total dose to point A and point F 35 - 45 Gy(i) and 38 - 50 Gy(i) respectively in 4 fractions. The EBRT was given to the whole pelvic field, with 6 MV or 8 MV X-ray, 2 Gy per fraction, 4 times per week. The total dose was 45 to 50 Gy (the field was blocked 4 cm after 20 - 30 Gy), the total treatment time was 5 - 6 weeks. RESULTS: The follow-up time was 36 - 96 months, with an average of 42 months. The three year local control and overall survival rate was 88% (35/40) and 75% (30/40) respectively for all patients. Of those patients of stage Ib, they were 93% (14/15) and 87% (13/15), respectively, higher than stage II [80% (12/15), 87% (13/15); P > 0.05], significantly higher than stage III, IV [60% (6/10), 50% (5/10); P < 0.01]. Three year local control and overall survival rate of G(1) grade was 92% (23/25) and 88% (22/25) respectively, significantly higher than G(2) - G(3) grade [80% (12/15), 53% (8/15); P < 0.01]. Three year local control and overall survival rate of adenocarcinoma was 93% (28/30) and 87% (26/30) respectively, significantly higher than squamous adenocarcinoma and papillary adenocarcinoma [70% (7/10), 30% (3/10); P < 0.01]. The grade 2 late radiation cystitis was 2% (1/40), and grade 2, 3 radiation proctitis and sigmoiditis were 10% (4/40). CONCLUSIONS: Combined (252)Cf ICBT and EBRT may be safe and effective for advanced endometrial cancer. The most important prognostic factors were stage, pathological type and differentiation of endometrial cancer.

[Combination of external beam radiotherapy and 252Cf neutron intracavitary brachytherapy for cervical cancer].

OBJECTIVE: To evaluate the curative effect of 252Cf brachytherapy on advanced cervical cancers. METHODS: A total of 77 cervical cancer patients were staged into IIa-IVa according to the standard of International Federation of Gynecology and Obstetrics (FIGO), including 13 cases of stage IIa, 32 of stage IIb, 18 of stage IIIa, 10 of stage IIIb, and 4 of stage IVa. 252Cf brachytherapy was delivered at 8-11 Gy/fraction per week, the total dose of reference point A was 36-45 Gy in 4-5 fractions. The whole pelvic cavity was irradiated with 6 MV X-ray, 200 cGy/fraction, 4 times per week. The total dose of external beam radiotherapy was 45-50 Gy (the center of whole pelvic field was blocked by 4 cm in width after 20-36 Gy), the total treatment course was 5-6 weeks. The 3-year survival rate and late complication rate in bladder and rectum were evaluated. RESULTS: The local control rate of all patients after 3-year was 94% (72/77); the overall survival rate was 82% (63/77). In particular, the 3-year survival rate was 85% (11/13) for stage IIa, 94% (30/32) for stage IIb, 78% (14/18) for stage IIIa, 70% (7/10) for stage IIIb, 25% for stage IVa cancers. The late complication rate was 3% for cystitis, 5% for proctitis. CONCLUSIONS: Combination of 252Cf brachytherapy and external beam radiotherapy can be well-tolerated by cervical cancer patients. The 3-year survival rate for stage II and III patients is high and late complication rate in bladder and rectum is low.