Alternative RNA splicing defects in pediatric cancers: new insights in tumorigenesis and potential therapeutic vulnerabilities.

BACKGROUND: Compared with adult cancers, pediatric cancers are uniquely characterized by a genomically stable landscape and lower tumor mutational burden. Alternative splicing, however, a global cellular process that produces different messenger RNA/protein isoforms from a single messenger RNA transcript, has been increasingly implicated in the development of pediatric cancers. DESIGN: We review the current literature on the role of alternative splicing in adult cancer, cancer predisposition syndromes, and pediatric cancers. We also describe multiple splice variants identified in adult cancers and confirmed through comprehensive genomic profiling in our institutional cohort of rare, refractory, and relapsed pediatric and adolescent young adult cancer patients. Finally, we summarize the contributions of alternative splicing events to neoantigens and chemoresistance and prospects for splicing-based therapies. RESULTS: Published dysregulated splicing events can be categorized as exon inclusion, exon exclusion, splicing factor up-regulation, or splice site alterations. We observe these phenomena in cancer predisposition syndromes (Lynch syndrome, Li-Fraumeni syndrome, CHEK2) and pediatric leukemia (B-cell acute lymphoblastic leukemia), sarcomas (Ewing sarcoma, rhabdomyosarcoma, osteosarcoma), retinoblastoma, Wilms' tumor, and neuroblastoma. Within our institutional cohort, we demonstrate splice variants in key regulatory genes (CHEK2, TP53, PIK3R1, MDM2, KDM6A, NF1) that resulted in exon exclusion or splice site alterations, which were predicted to impact functional protein expression and promote tumorigenesis. Differentially spliced isoforms and splicing proteins also impact neoantigen creation and treatment resistance, such as imatinib or glucocorticoid regimens. Additionally, splice-altering strategies with the potential to change the therapeutic landscape of pediatric cancers include antisense oligonucleotides, adeno-associated virus gene transfers, and small molecule inhibitors. CONCLUSIONS: Alternative splicing plays a critical role in the formation and growth of pediatric cancers, and our institutional cohort confirms and highlights the broad spectrum of affected genes in a variety of cancers. Further studies that elucidate the mechanisms of disease-inducing splicing events will contribute toward the development of novel therapeutics.

[Clinical analysis of 554 patients with colorectal diverticulosis].

Objective: Most patients with asymptomatic colorectal diverticulosis are easily overlooked. However, some of diverticulosis become diverticulitis, bleeding and even perforation, which cause extensive harm to patients. The purpose of this study is to analyze the incidence, clinical features, diagnosis and treatment of colorectal diverticulosis in order to improve the clinical understanding of diverticulosis and its related complications. Methods: A descriptive cohort study was carried out. Clinical data of 554 patients with colorectal diverticulosis confirmed by CT, colonoscopy, digestive tract radiography or operation in Peking University First Hospital from January 2009 to June 2019 were retrospectively analyzed. Patients with malignant tumors, autoimmune diseases, long term use of immunosuppressive drugs, chronic liver diseases and renal diseases, and mental disorders were excluded. The analysis parameters included gender, onset age, clinical symptoms, location of diverticulitis, treatment and prognosis. According to the criteria established by the World Society of Emergency Surgery (WSES), acute diverticulitis was divided into 5 stages based on the extension of the infectious process. Stage 0 was simple diverticulitis and stage 1-4 was complicated diverticulitis. Results: Among the 554 patients with colorectal diverticulosis, 358 (64.6%) were males, the median onset age was 63 years; 191 patients (34.5%) had various digestive symptoms, of whom 113 (20.4%) had chronic constipation and abdominal distension, 78 (14.1%) had chronic diarrhea and abdominal pain; the other 363 patients had no obvious abdominal symptoms. Four hundred and six patients were found by colonoscopy and 465 patients were found by CT. Twenty-five patients were diagnosed by lower gastrointestinal tract radiography and 3 were confirmed during operation. There were 339 patients with multiple diverticula (61.2%) and 215 patients with single diverticulum (38.8%). 76.5% (424/554) of diverticula were located in colon, 37.0% (205/554) in ascending colon, 21.3% (118/554) in multiple sites, and 2.2% (12/554) in rectum. The median diameter of diverticulum was 7 mm, and 78 cases (14.1%) was ≥30 mm. Forty-nine patients (8.8%) developed acute diverticulitis, including 13 patients with simple diverticulitis and 36 patients with complicated diverticulitis. Among 36 patients with complicated diverticulitis, 29 (80.6%) were males, 27 (75.0%) had recurrent abdominal pain and fever before onset; diverticula of 25 cases were located in sigmoid colon; 11 cases in ascending colon. Nine cases developed sigmoid colon perforation and 8 cases developed vesicocolonic fistula, and these 17 patients underwent surgical treatment. The other 19 cases with complicated diverticulitis developed gastrointestinal bleeding, of whom 18 cases were male, 11 cases were located in ascending colon; 13 cases were healed after conservative treatment, 4 cases received endoscopic hemostatic intervention, and 2 cases underwent surgery. Conclusions: Colorectal diverticulosis is more common in male patients, and CT and colonoscopy are main diagnostic methods. The symptoms of complicated colonic diverticulitis are related to the location of diverticulum. In addition to symptomatic treatment, surgical procedures are the most important treatments.

[Clinical research progress on disappearing colorectal liver metastases].

Colorectal cancer is currently one of the most common digestive system tumors, and the liver is the most common metastatic site of colorectal cancer. In recent years, with the continuous development of the multidisciplinary treatment for colorectal cancer patients, there are quite a few cases of disappearing liver metastases (DLM) after receiving preoperative chemotherapy (or combined targeted drug therapy), and the diagnosis and treatment of DLM is currently still a very challenging and controversial topic. This article sorts out the related researches on DLM in recent years, mainly including the following 4 aspects: (1) The factors associated with DLM, including the size and number of liver metastases, chemotherapy regimens and cycles, targeted therapy drugs, and the pattern of liver metastases, Ras/Braf status and the location of the primary lesion. (2) The relationship between DLM and true complete response (pathological complete response and persistent clinical complete response), and the related predictive factors of pathological complete response. (3) Clinical evaluation of DLM: preoperative evaluation includes ultrasound, CT, MRI, and PET, while intraoperative evaluation includes intraoperative exploration, intraoperative ultrasound, and augmented reality. (4) DLM treatment strategies, including surgical treatment, local treatment, non-surgical treatment and individualized treatment.

[Research progress of conversion therapy in colorectal cancer liver metastases].

The probability of developing liver metastases in patients with colorectal cancer is 40%-50%. Liver metastases remain an important adverse factor affecting long-term prognosis of colorectal cancer patients. Surgical resection of liver metastases is the only potentially curative treatment option. After comprehensive treatment, initially unresectable liver metastases might be converted to resectable tumors. This concept is known as conversion therapy. In this review, research status of conversion therapy in colorectal cancer liver metastases was summarized, providing updated concept of resectability, discussions on the assessment of tumor response and timing of operation, debates on the influence on tumor sidedness, and latest advancement in the treatment strategy of conversion therapy. Through analyzing existing problems, we hope to offer insights into possible progress in the future and provide references for the development of clinical practice.

TGF-ß1 aggravates degenerative nucleus pulposus cells inflammation and fibrosis through the upregulation of angiopoietin-like protein 2 expression.

OBJECTIVE: Inflammation and fibrosis progress of nucleus pulposus (NP) cells participate in the pathologic changes of intervertebral disc degeneration (IDD). ANGPTL2 is well known for its angiogenesis and proinflammatory properties and transforming growth factor ß1 (TGF-ß1) is also responsible for tissue fibrosis. However, the role of ANGPTL2 in IDD and whether it is related to TGF-ß1 remains unclear. This study aims to explore the relation of TGF-ß1 and ANGPTL2 in the degenerative process of NP cells. PATIENTS AND METHODS: We isolated NP cells of NP tissues provided from the spine fracture patients. IL-1ß was used to induce the NP cells degeneration. To determine the effect of TGF-ß1 and ANGPTL2 on NP cell degeneration, we regulated the cellular TGF-ß1 and ANGPTL2 expression by Recombinant human protein stimulation and siRNA transfection. Quantitative real-time polymerase chain reaction (qRT-PCR) or Western blot was employed to investigate the expression of TGF-ß1, ANGPTL2, IL-6, TNF-α, collagen I, and collagen III. RESULTS: TGF-ß1 overexpression aggravated the ANGPTL2, IL-6, TNF-α, collagen I, and collagen III expressions of NP cells that caused by IL-1ß, which was rejected by ANGPTL2 gene silencing. Besides, the silencing of TGF-ß1 weakened the ANGPTL2 expression. ANGPTL2 overexpression promoted the NP cells inflammation and fibrosis via increasing IL-6, TNF-α, collagen I, and collagen III expression, which was sharpened by a consequent increase of TGF-ß1 expression. CONCLUSIONS: This study, for the first time, points that TGF-ß1 aggravates degenerative NP cells inflammation and fibrosis via the mediation of ANGPTL2.

[Analysis on risk factors of the degree of radical resection and prognosis of patients with locally recurrent rectal cancer].

Objective: To evaluate the factors affecting the degree of radical resection and the prognosis of patients with locally recurrent rectal cancer (LRRC). Methods: A retrospective case-control study was performed. Clinical data of 111 patients with LRRC undergoing operation at the General Surgery Department of Peking University First Hospital from January 2009 to August 2019 were analyzed retrospectively. The "Peking University First Hospital F typing" was performed according to the preoperative images of the pelvic involvement. The pelvis was assigned into four directions: the front wall, lateral sides of the pelvic wall and the sacrum. According to the degree of pelvic wall involvement, F typing included F0 type (no involvement of the pelvic wall, the cancer only involved the adjacent organs or invaded conteriorly the urinary tract, genital organs or small intestine), F1 type (cancer involved the pelvic wall in one direction, such as the sacrum, or one side of the pelvic wall), F2 type (cancer involved the pelvic wall in two directions) and F3 type (cancer involved the pelvic wall in three directions). Case inclusion criteria: (1) LRRC was confirmed by imaging and pathological examination of samples (puncture or endoscopic biopsy); (2) complete clinical and follow-up data; (3) informed consent of patient. Those with dysfunction of heart, lung, etc., intolerance of operation, F3 type indicated by image, and distant metastasis were excluded. The degree of radical resection was evaluated according to the postoperative pathological results. Patients were followed up every 12 months and related examinations were arranged. The univariate analysis of radical resection was performed by χ(2) test, and the multivariate analysis was performed by logistic methods. The survival rate was calculated by Kaplan-Meier method and the survival curve was drawn. The survival rate was compared by log-rank test. Cox proportional hazards model was used to analyze the factors affecting the prognosis of patients with LRRC. Results: A total of 111 patients were included in this study. Of 111 patients, 59 were male and 52 were female; recurrent age of 36 cases was ≥ 65 years old; CEA level of 48 cases was ≥15 µg/L. According to the "Peking University First Hospital F typing", 70 cases were F0 type, 38 F1 type and 3 F2 type. Surgical procedures were abdominoperineal resection (n=28), posterior pelvic exenteration (n=32), and total pelvic exenteration (n=51, including 1 case of TPE combined with sacrectomy). According to the postoperative pathological results, R0, R1 and R2 resections were 83, 20 and 8 cases, respectively. Univariate analysis showed that the degree of radical resection was associated with the secondary surgical procedure, F typing and lymph node metastasis (all P<0.05). Multivariate analysis showed that F typing (F1-F2) was an independent risk factor for non- R0 resection (OR=37.256, 95%CI:8.572 to 161.912, P<0.001). The morbidity of operative complications was 22.5% (25/111); the perioperative mortality was 1.8% (2/111); the local recurrence rate after the second operation was 37.8% (42/111). The 3- and 5-year overall survival rates were 41.2% and 21.9% respectively. The 3-year survival rates of patients with and without postoperative chemotherapy were 52.7% and 32.4% respectively (P=0.005). The 3-year survival rates of patients with lower (<15 µg/L) and higher CEA level (≥15 µg/L) were 52.9% and 24.3% respectively (P<0.001). The 3-year survival rates of patients with R0, R1 and R2 resection were 49.8%, 21.3% and 8.5% respectively (P=0.002). The 3-year survival rates of patients with F0, F1 and F2 type were 52.7%, 22.0% and 0 respectively (P<0.001). Cox analysis confirmed that the degree of radical resection (HR=2.088, 95%CI:1.095 to 3.979, P=0.025), the CEA level before the secondary operation (HR=1.857, 95%CI:1.157 to 2.980, P=0.010) and postoperative chemotherapy (HR=1.826, 95%CI:1.137 to 2.934, P=0.013) were independent factors affecting the prognosis. Conclusions: The indication of LRRC surgical treatments must be strictly limited. Evaluation of the fixation site to the pelvic wall is helpful for improving the rate of R0 resection. Lower preoperative CEA level, radical resection and postoperative chemotherapy are protective factors of prolonged overall survival time of patients with LRRC.

[Effects of different methods on drying root canal by near-field microwave detection system].

OBJECTIVE: To set up near-field microwave detection system, to evaluate the accuracy of measuring water and to compare the effects of different methods on drying root canal. METHODS: Known and quantitative distilled water in Eppendorf tube and root canal was detected by near-field microwave detection system which was set up first. The microwave reflection coefficient was recorded so as to evaluate the accuracy of the measuring water. 12 single-rooted mandibular premolar teeth with apical matureness and a curvature less than 10° were decoronated, leaving roots 14 mm in length. After instrumentation to F3, the root canals were air-dried and injected with 10 µL distilled water each. Then all the root canals were assigned to 4 groups (n=12) according to different methods on root canal drying. Near-field microwave detection system was used to detect the change of water after air-drying and root canal drying so as to evaluate the effects of different root canal drying methods: (1) negative control group, the canals were injected with 10 µL distilled water and no root canal drying method was performed; (2) paper points drying experimental group, four #60 paper points were used to blot-dry the middle and upper part of the canal, followed by #40 paper points blot-drying the apical area. The above step was repeated with the tip of paper point that appeared with no water under microscope as finished; (3) 95% (volume fraction) ethanol drying experimental group, after the distilled water was removed with paper points (similar to paper points experimental group), 10 µL 95% ethanol was injected into the canal and was left in place for 10 s, then paper points were used to dry the canal; (4) 70% (volume fraction) isopropyl alcohol drying experimental group, after the distilled water was removed with paper points (similar to paper points experimental group), 10 µL 70% isopropyl alcohol was injected into the canal and was left in place for 10 s, then paper points were used to dry the canal. The linear correlation analysis was used to test the accuracy of the measuring water by near-field microwave detection method and physical measurement. The Pearson correlation coefficient was calculated based on the data. The effects of different methods on drying root canal were analyzed by Chi square test. RESULTS: The R2 for the microwave reflection coefficient and the physical measurement of the water in Eppendorf tube and root canal were 0.991 and 0.970. The microwave reflection coefficient in all the experimental groups showed significant difference (P<0.05). After drying with paper points, the residual water was the most, and the microwave reflection coefficient was the highest: 0.023. While after 70% isopropyl alcohol drying root canal, the residual water was the least with the microwave reflection coefficient 0.006, showing that the effect of drying was the best. CONCLUSION: Near-field microwave detection system could detect the change of water in root canal. The effect of root canal drying by 70% isopropyl alcohol was superior to 95% ethanol and paper points.

[Preoperative evaluation using multi-slice spiral CT angiography of right-side colon vascular in laparoscopic radical operation for right colon cancer].

Objectives: To examine the value of multi-slice spiral CT angiography (MSCTA) in the analysis of anatomical variation and structural classification of right colon vessels. Methods: From August 2015 to August 2017, 198 patients (96 of whom underwent laparoscopic radical resection of right colon cancer) at Department of General surgery of Peking University First Hospital were retrospectively collected, and the results of abdominal enhanced CT scan were collected and three-dimensional reconstruction of blood vessels was performed. There were 104 males and 94 females. The age was 64(27) years (M(Q(R)), range: 19 to 87 years). Right gastroepiploic vein, anterior superior pancreaticoduodenal vein, right colonic vein (RCV), superior right colonic vein, ileocolon artery or vein (ICA or ICV), middle colon artery or vein (MCA or MCV) and Henle trunk were observed and recorded respectively. The anatomical relationship between the positions of blood vessels, the length of Henle trunk and surgical trunk were measured. Results: ICV and ICA were the most constant anatomic structures. The ICV/ICA of all patients came directly from SMV/SMA, 36.9% (73/198) ICV going in front of SMV and 63.1% (125/198) behind SMV. 72.2% (143/198) of the patients had RCV imported into Henle trunk and the rest into SMV. Middle colonic vein (MCV) could be observed in 81.3% (161/198) of the cases. 81.4% (131/161) of MCV were imported into SMV, 16.8% (27/161) into Henle trunk, 1.2% (2/161) into the first jejunal vein and 0.6% (1/161) into the splenic vein. Henle trunk was divided into 4 types, among which the occurrence probability of gastric node and pancreatic trunk was the highest. The dry length of Henle trunk was (0.82±0.39) cm (range: 0.37 to 1.68 cm). The length of surgical trunk was (2.54±0.83) cm (range: 1.57 to 3.95 cm). Accuracy of MSCTA results was 96.9%(93/96). Conclusions: Anatomical variation of blood vessels in the right colon is common. Abdominal CT angiography can accurately determine the anatomical structure of the blood vessels in the right colon.

[Progress of molecular subtypes of colorectal cancer].

Colorectal cancer is one of the leading causes of morbidity and mortality around the world. Although the staging and classification systems, such as tumor/node/metastasis (TNM) staging, are widely used in clinic, there are some limitations. For example, the patients with the same pathological type, TNM stage and treatment regimen show a completely different prognosis and outcome. In the present, molecular subtyping was concerned for a more precise and accurate staging of colorectal cancer. Herein, we reviewed the literature of the molecular subtypes of colorectal cancer in the past decades. The clinical significance of various molecular subtyping systems was evaluated and compared. It will provide reference for the precision medicine of colorectal cancer in the future.

Neuroblastomas vary widely in their sensitivities to herpes simplex virotherapy unrelated to virus receptors and susceptibility.

Although most high-risk neuroblastomas are responsive to chemotherapy, relapse is common and long-term survival is < 40%, underscoring the need for more effective treatments. We evaluated the responsiveness of 12 neuroblastoma cell lines to the Δγ134.5 attenuated oncolytic herpes simplex virus (oHSV), Seprehvir (HSV1716), which is currently used in pediatric phase I trials. We found that entry of Seprehvir in neuroblastoma cells is independent of the expression of nectin-1 and the sum of all four known major HSV entry receptors. We observed varying levels of sensitivity and permissivity to Seprehvir, suggesting that the cellular anti-viral response, not virus entry, is the key determinant of efficacy with this virus. In vivo, we found significant anti-tumor efficacy following Seprehvir treatment, which ranged from 6/10 complete responses in the CHP-134 model to a mild prolonged median survival in the SK-N-AS model. Taken together, these data suggest that anti-tumor efficacy cannot be solely predicted based on in vitro response. Whether or not this discordance holds true for other viruses or tumor types is unknown. Our results also suggest that profiling the expression of known viral entry receptors on neuroblastoma cells may not be entirely predictive of their susceptibility to Seprehvir therapy.

Expression of HSV-1 receptors in EBV-associated lymphoproliferative disease determines susceptibility to oncolytic HSV.

Epstein-Barr virus (EBV)-associated B-cell lymphoproliferative disease (LPD) after hematopoietic stem cell or solid organ transplantation remains a life-threatening complication. Expression of the virus-encoded gene product, EBER, has been shown to prevent apoptosis via blockade of PKR activation. As PKR is a major cellular defense against Herpes simplex virus (HSV), and oncolytic HSV-1 (oHSV) mutants have shown promising antitumor efficacy in preclinical models, we sought to determine whether EBV-LPD cells are susceptible to infection by oHSVs. We tested three primary EBV-infected lymphocyte cell cultures from neuroblastoma (NB) patients as models of naturally acquired EBV-LPD. NB12 was the most susceptible, NB122R was intermediate and NB88R2 was essentially resistant. Despite EBER expression, PKR was activated by oHSV infection. Susceptibility to oHSV correlated with the expression of the HSV receptor, nectin-1. The resistance of NB88R2 was reversed by exogenous nectin-1 expression, whereas downregulation of nectin-1 on NB12 decreased viral entry. Xenografts derived from the EBV-LPDs exhibited only mild (NB12) or no (NB88R2) response to oHSV injection, compared with a NB cell line that showed a significant response. We conclude that EBV-LPDs are relatively resistant to oHSV virotherapy, in some cases, due to low virus receptor expression but also due to intact antiviral PKR signaling.

[Inhibiting gene expression in vivo by virus-mediated small interfering RNA].

Inhibiting gene expression in specific tissues and organs through intravenous injection would be the ultimately preferred method of disease therapy. Here, we report the successful delivery of lentivirus-mediated small interfering RNA (siRNA) to suppress the GFP gene expression in living mice. First, a lentiviral vector with siRNA (len-siRNA) driven by H1 promoter was constructed to suppress GFP expression effectively in Mel cells. When the len-siRNA virus was injected into transgenic mice, the GFP expression was significantly suppressed (over 15% reduction) in the recipient mice compared to the control mice and the suppressing effect lasted more than one week after injection. Our results demonstrate a new effective approach to inhibit gene expression by siRNA and lentiviral vectors. Further development of this suppression of gene expression siRNA drug should result in applications not only for cancers but also for infectious and immune diseases.

WITHDRAWN: A radio frequency biosensor with gold nanoparticle probes.

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Temporal lobe magnetic resonance spectroscopic imaging following selective amygdalohippocampectomy for treatment-resistant epilepsy.

OBJECTIVES: Magnetic resonance spectroscopic imaging (MRSI) may show circumscribed or extensive decreased brain N-acetyl aspartate (NAA)/creatine and phosphocreatine (Cr) in epilepsy patients. We compared temporal lobe MRSI in patients seizure-free (SzF) or with persistent seizures (PSz) following selective amygdalohippocampectomy (SAH) for medically intractable mesial temporal lobe epilepsy (mTLE). We hypothesized that PSz patients had more extensive temporal lobe metabolite abnormalities than SzF patients. MATERIALS AND METHODS: MRSI was used to study six regions of interest (ROI) in the bilateral medial and lateral temporal lobes in 14 mTLE patients following SAH and 11 controls. RESULTS: PSz patients had more temporal lobe ROI with abnormally low NAA/Cr than SzF patients, including the unoperated hippocampus and ipsilateral lateral temporal lobe. CONCLUSION: Postoperative temporal lobe MRSI abnormalities are more extensive if surgical outcome following SAH is poor. MRSI may be a useful tool to improve selection of appropriate candidates for SAH by identifying patients requiring more intensive investigation prior to epilepsy surgery. Future prospective studies are needed to evaluate the utility of MRSI, a predictor of successful outcome following SAH.

Milk/dairy products consumption, galactose metabolism and ovarian cancer: meta-analysis of epidemiological studies.

Ovarian cancer is the fifth most common cause of cancer death among women and the leading cause of gynaecological cancer death in the United States. Milk/dairy products consumption was considered to be a risk factor for ovarian cancer mainly because milk carbohydrate-lactose and galactose metabolism is toxic to oocytes. However, recent evidence does not support this hypothesis completely. We collected epidemiological studies related to the association between milk/dairy products consumption or galactose metabolism (lactose, galactose, galactose-1-phosphate uridyltransferase, lactose/transferase) and ovarian cancer published between January 1966 and August 2003 and found 27 items from 22 independent studies. Twenty studies were case-control studies and the other two were cohort studies. A meta-analysis method was conducted to estimate relative risk combining all relative data. In general, we did not find any association between milk/dairy products or galactose metabolism and ovarian cancer risk in this meta-analysis. The consumption of whole milk and butter, which contain relatively high amounts of fat, was positively (relative risk > 1.2), but not significantly, associated with an increased risk.

The experience of Japan as a clue to the etiology of testicular and prostatic cancers.

In Japan dramatic lifestyle changes occurred after World War 2. To examine the experience of Japan as a clue to the etiology, trends in the mortality rates of testicular and prostatic cancers from 1947 to 1998 were related to changes in dietary practices. The male population born before 1945 had a peak in death from testicular cancer in their thirties or forties, whereas those born after 1946 had a peak in their twenties. The death rate of prostatic cancer increased 25-fold almost linearly after the war. The intake of milk, meat, and eggs increased 20-, 9-, and 7-fold, respectively, after the war. In connection with the development and growth of testicular and prostatic cancers in Japan, particular attention should be paid to milk, because the increase in its consumption in this country is a recent occurrence and because milk contains considerable amounts of estrogens plus saturated fats.

Is milk responsible for male reproductive disorders?

The role of environmental compounds with estrogenic activity in the development of male reproductive disorders has been a source of great concern. Among the routes of human exposure to estrogens, we are particularly concerned about cows' milk, which contains considerable amounts of estrogens. The major sources of animal-derived estrogens in the human diet are milk and dairy products, which account for 60-70% of the estrogens consumed. Humans consume milk obtained from heifers in the latter half of pregnancy, when the estrogen levels in cows are markedly elevated. The milk that we now consume may be quite unlike that consumed 100 years ago. Modern genetically-improved dairy cows, such as the Holstein, are usually fed a combination of grass and concentrates (grain/protein mixes and various by-products), allowing them to lactate during the latter half of pregnancy, even at 220 days of gestation. We hypothesize that milk is responsible, at least in part, for some male reproductive disorders.

Aggrecan domains expected to traffic through the exocytic pathway are misdirected to the nucleus.

In this article, we report the misdirected targeting of expressed aggrecan domains. Aggrecan, the chondroitin sulfate (CS) proteoglycan of cartilage, normally progresses through the exocytic pathway. Proteins expressed from constructs containing the putative aggrecan signal sequence (i.e., the first 23 N-terminal amino acids), specified globular (G) domains G1 and/or G3, and a segment of the CS domain were detected in the endoplasmic reticulum (ER) and Golgi complex. Although proteins expressed from constructs containing the putative signal and G3, but lacking G1, were detected to a limited extent in the secretory pathway, they primarily accumulated in nuclei. Discrete nuclear inclusions were seen when G3 was expressed. Immunoelectron microscopic characterization of the inclusions suggested the association of nuclear G3 with other proteins. When signal-free G3 constructs and those with G3 immediately following the N-terminal signal were expressed, abundant dispersed accumulations filled the nucleoplasm. The data suggest first, that signal-free and signal-containing G3 proteins enter the nucleus from the cytosol, and second, that the entry of signal-containing G3 proteins into the ER lumen is inefficient. Hsp25, Hsp70, and ubiquitin were colocalized with nuclear G3, indicating the involvement of chaperones and the degradative machinery in the formation and/or attempted disposal of the abnormal nuclear inclusions. Overall, the results focus attention on (1) intracellular protein trafficking at the ER membrane and the nuclear envelope and (2) chaperone interactions and mechanisms leading to abnormal protein deposition in the nucleus.